Localization and Characterization of Flavivirus Envelope Gylcoprotein Cross-Reactive Epitopes and Methods for their Use

ABSTRACT

Disclosed herein is a method for identifying flavivirus cross-reactive epitopes. Also provided are flavivirus E-glyco-protein cross-reactive epitopes and flavivirus E-glycoprotein crossreactive epitopes having reduced or ablated cross-reactivity (and polypeptides comprising such epitopes), as well as methods of using these molecules to elicit an immune response against a flavivirus and to detect a flaviviral infection.

PRIORITY CLAIM

This application claims the benefit of U.S. Provisional PatentApplication No. 60/591,898 filed Jul. 27, 2004, which is incorporatedherein by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made by the Centers for Disease Control andPrevention, an agency of the United States Government. Therefore, theU.S. Government has certain rights in this invention.

FIELD

This disclosure relates to a structure-based rational mutagenesis methodfor identifying flavivirus envelope (E)-glycoprotein cross-reactiveepitopes. The disclosure further relates to flavivirus E-glycoproteincross-reactive epitopes and mutants thereof having reduced or ablatedcross-reactivity. Flavivirus cross-reactive E-glycoprotein epitopes withreduced or ablated cross-reactivity are useful in the diagnosis,inhibition and treatment of diseases caused by flaviviruses.

BACKGROUND

The Flaviviridae are a diverse family of enveloped viruses infectingboth arthropods and vertebrates. Flaviviruses have a positive-sensesingle-stranded RNA genome 10.7 kb in length, transcribed into a singlepolyprotein precursor encoding three structural proteins, capsid,premembrane (prM), envelope (E), and seven non-structural proteins(Lindenbach & Rice, Flaviviridae: the viruses and their replication. InFields Virology, 4^(th) ed., Knipe and Howley. Eds., Philadelphia,Lippincott Williams & Wilkins, pp. 991-1041, 2001; Rice et al., Science229:726-33, 1985). The flavivirus E-glycoprotein is the primary antigen,inducing protective immunity; it is essential for membrane fusion, andmediates binding to cellular receptors (Allison et al., J. Virol.75:4268-75, 2001; Crill & Roehrig, J. Virol. 75:7769-73, 2001; Rey etal., Nature 375:291-98, 1995). Flavivirus E-glycoprotein thereforedirectly affects host range, tissue tropism, and the virulence of theseviruses.

The flavivirus E-glycoprotein contains three structural and functionaldomains. Domain 1 (DI) is an 8-stranded β-barrel containing two largeinsertion loops that form the elongated finger-like domain II (DII) (Reyet al., Nature 375:291-98, 1995). DII is involved in stabilizing theE-glycoprotein dimer and contains the internal fusion peptide (Allisonet al., J. Virol. 75:4268-75, 2001). Domain III (DIII) forms aten-stranded β-barrel with an immunoglobulin-like fold and contains thecellular receptor-binding motifs (Crill & Roehrig, J. Virol. 75:7769-73,2001; Modis et al., PNAS 100:6986-91, 2003). DI and DIII containpredominately type-specific and subcomplex-reactive epitopes, whereasDII contains the major flavivirus group- and subgroup-cross-reactiveepitopes, which are sensitive to reduction and denaturation and areformed from discontinuous amino acid sequences (Mandl et al., J. Virol.63:564-71, 1989; Rey et al., Nature 375:291-98, 1995; Roehrig et al.,Virology 246:317-28, 1998).

Members of the Flaviviridae family that infect humans frequently causesevere morbidity and mortality, and epidemics of flaviviruses continueto be a major public health concern worldwide. More than two billionpeople are at risk of being infected with members of the genusFlavivirus which includes at least 70 distinct virus species (Burke &Monath, Flaviviruses. In Fields Virology, 4^(th) ed., Knipe and Howley.Eds., Philadelphia, Lippincott Williams & Wilkins, pp. 1043-1125, 2001;Kuno et al., J. Virol. 72:73-83, 1998; Solomon & Mallewa, J. Infect.42:104-15, 2001). The medically important flaviviruses include yellowfever (YF) virus in Africa, Latin and South America; Japaneseencephalitis (JE) virus in Asia and Australia; West Nile (WN) virus inAfrica, Central Europe, and most recently in North America; tick-borneencephalitis (TBE) complex viruses in the temperate regions of Europe,North America and Asia; and the four serotypes of dengue viruses (DEN-1,-2, -3, and -4) in tropical and subtropical regions of the world(Lindenbach & Rice, Flaviviridae: the viruses and their replication. InFields Virology, 4^(th) ed., Knipe and Howley. Eds., Philadelphia,Lippincott Williams & Wilkins, pp. 991-1041, 2001).

Human infection by flaviviruses results in a humoral immune responseinvolving virus species-specific as well as flavivirus cross-reactiveantibodies (Calisher et al., J. Gen. Virol 70:37-43, 1989; Tesh et al.,Emerg. Inf. Dis. 8:245-51, 2002). The presence of flaviviruscross-reactive antibodies in human sera produces two public healthconcerns upon secondary infection with a heterologous flavivirus.Serodiagnosis of secondary flavivirus infections, especially in areaswith multiple co-circulating flaviviruses, can be particularly difficultdue to the inability to differentiate primary from secondarycross-reactive serum antibodies using currently available viralantigens. Therefore, definitive epidemiological information eithercannot be obtained or is delayed to the point that effective control andprevention strategies may be delayed. Additionally, the presence ofsub-neutralizing levels of flavivirus cross-reactive serum antibodiesmay result in increasing the severity of secondary flavivirus infectionsdue to antibody-dependant enhancement (ADE), in particular, followingsecondary dengue virus infection (Ferguson et al., PNAS 96:790-94, 1999;Halstead, Rev. Infect. Dis. 11:830-39, 1989; Takada & Kawaoka, Rev. Med.Virol. 13:387-98, 2003; Wallace et al., J. Gen Virol. 84:1723-28, 2003).Thus, there exists a need for a method for identifying andcharacterizing flavivirus cross-reactive epitopes for improvedflavivirus serodiagnosis and development of flavivirus vaccines.

SUMMARY OF THE DISCLOSURE

Multiple flavivirus E-glycoprotein cross-reactive epitopes and mutantE-glycoprotein polypeptides thereof exhibiting reduced or ablatedcross-reactivity have been identified. In various embodiments, theseE-glycoprotein polypeptides with reduced or ablated cross-reactivity arecapable of eliciting effective type-specific immune responses againstflaviviruses. In one example, the identified cross-reactive epitopesincorporate the highly conserved Gly₁₀₄, Gly₁₀₆, and Leu₁₀₇ residues. Inanother example, the identified cross-reactive epitope centers on thestrictly conserved Trp₂₃₁ residue and its structurally related neighborsGlu₁₂₆ and Thr₂₂₆.

Also described herein are recombinant flavivirus E-glycoproteinconstructs that can be used directly or indirectly to stimulateflavivirus type-specific antibodies. These constructs are designed toelicit T-cell, B-cell, or both T-cell and B-cell responses againstflavivirus type-specific epitopes. The constructs, when integrated intoa vector, can be used as immunogens, can be used as DNA vaccines, andcan be used as sources of recombinant protein for stimulation of immuneresponses in subjects, as well as for protein boosts to subjects whohave received a nucleic acid construct previously. Also provided aremethods of identifying and characterizing flavivirus E-glycoproteinamino acid residues incorporated into cross-reactive epitopes, usingstructure-based rational mutagenesis.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagrammatical representation of the quaternary structure ofthe DEN-2 virus E-glycoprotein homodimer, top view, looking down towardsthe viral surface, showing the locations of flavivirus cross-reactiveepitope residues (space-filling representation). The structural andfunctional domains I, II, and III are also shown.

FIG. 2 is a series of diagrammatical representations of the structurallocations of cross-reactive epitope residues for flaviviruscross-reactive monoclonal antibodies (mAbs) in the atomic structure ofthe DEN-2 virus E-glycoprotein dimer, as well as a bar graph indicatingfold reductions in mAb reactivities assayed by indirectimmuno-fluorescence assay (IFA) and/or antigen-capture ELISA (Ag-ELISA)for mutations at these E-glycoprotein positions.

FIG. 2A is a diagrammatical representation of a portion of the atomicstructure of the DEN-2 virus E-glycoprotein homodimer, showing theflavivirus group-reactive mAb 4G2 and 6B6C-1 epitope residues from thefusion peptide region of DII. The flavivirus fusion peptide comprisesthe highly conserved E-glycoprotein residues 98-113, which form asurface exposed loop of hydrophobic residues rich in glycine at the tipof DII (Rey et al., Nature 375:291-98, 1995; Allison et al., J. Virol.75:4268-75, 2001). The view is looking downward toward the viralmembrane surface at an angle of approximately 45°, while looking intowards the fusion peptide region about 45° off of parallel to thedimer's longitudinal axis. The molecular surfaces of DI and DIII fromthe alternate sub-unit are depicted as space-filling Van der Waalssurfaces to highlight the close fitting of the fusion peptide into thisregion. Fusion peptide residues 100-108 are depicted as stickrepresentations with the participating amino acids labeled. Glycanmoieties attached to Asn₁₅₃ and Asn₆₇ are labeled CHO153 and CHO67,respectively.

FIG. 2B is a diagrammatical representation of a portion of the atomicstructure of the DEN-2 virus E-glycoprotein homodimer, showing theflavivirus subgroup-reactive mAb 1B7-5 epitope residues. The view andlabeling are the same as in FIG. 2A. Identified residues are depicted assticks and labeled.

FIG. 2C is a bar graph showing fold decreases in mAb reactivities inAg-ELISA for DEN-2 VLPs with substitutions at the listed residues. mAbs4G2 and 6B6C-1 are flavivirus group-reactive and 1B7-5 is flavivirussubgroup-reactive. Substitutions at G₁₀₄ and W₂₃₁ produced plasmids thatwere unable to secrete measurable VLP antigen into tissue culture media.Therefore, fold decreases in mAb reactivities for these two constructsare from IFA. Wild-type plasmid did not produce an endpoint nearly asfar out in IFA as in Ag-ELISA (see Table 3), therefore the foldreductions for substitutions at G₁₀₄ and W₂₃₁ were not as great as forother constructs measured by Ag-ELISA, even though substitutions atthese two positions appeared to completely ablate mAb reactivity.

FIG. 3 is a bar graph showing the percent of cross-reactive epitoperesidue substitutions altering reactivities of mAbs of differentcross-reactivities. The total number of SLEV and WNV mAbs of each typeis shown in the legend on the y-axis, and the number of substitutionsaltering these mAbs is shown in the columns.

BRIEF DESCRIPTION OF THE APPENDICES

Appendix 1 contains Tables 1-13.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. In the accompanying sequence listing:

SEQ ID NOs: 1-12 show the nucleic acid sequences of mutagenic primersused to generate the K₆₄N mutation, T₇₆M mutation, Q₇₇R mutation, G₁₀₄Hmutation, G₁₀₆Q mutation, L₁₀₇K mutation, E₁₂₆A mutation, T₂₂₆Nmutation, W₂₃₁F mutation, W₂₃₁L mutation, H₂₄₄R mutation, and K₂₄₇Rmutation, respectively, in the pCB8D2-2J-2-9-1 DEN-2 prM/E expressionplasmid.

SEQ ID NOs: 13 and 14 show the nucleic and amino acid sequences of arecombinant DEN-2 virus E-glycoprotein antigen.

SEQ ID NOs: 15 and 16 show the nucleic and amino acid sequences of arecombinant DEN-2 virus E-glycoprotein antigen incorporating the G₁₀₄Hsubstitution.

SEQ ID NOs: 17 and 18 show the nucleic and amino acid sequences of arecombinant DEN-2 virus E-glycoprotein antigen incorporating the G₁₀₆Qsubstitution.

SEQ ID NOs: 19 and 20 show the nucleic and amino acid sequences of arecombinant DEN-2 virus E-glycoprotein antigen incorporating the L₁₀₇Ksubstitution.

SEQ ID NOs: 21 and 22 show the nucleic and amino acid sequences of arecombinant DEN-2 virus E-glycoprotein antigen incorporating the E₁₂₆Asubstitution.

SEQ ID NOs: 23 and 24 show the nucleic and amino acid sequences of arecombinant DEN-2 virus E-glycoprotein antigen incorporating the T₂₂₆Nsubstitution.

SEQ ID NOs: 25 and 26 show the nucleic and amino acid sequences of arecombinant DEN-2 virus E-glycoprotein antigen incorporating the W₂₃₁Fsubstitution.

SEQ ID NOs: 27 and 28 show the nucleic and amino acid sequences of arecombinant DEN-2 virus E-glycoprotein antigen incorporating the W₂₃₁Lsubstitution.

SEQ ID NOs: 29 and 30 show the nucleic and amino acid sequences of arecombinant DEN-2 virus E-glycoprotein antigen incorporating the doubleE₁₂₆A/T₂₂₆N substitution.

SEQ ID NOs: 31-79 show the nucleic acid sequences of mutagenic primersused to generate site-specific mutations into the SLEV and WNV E genes.

SEQ ID NOs: 80 and 81 show the nucleic and amino acid sequences of arecombinant SLEV virus E-glycoprotein antigen.

SEQ ID NOs: 82 and 83 show the nucleic and amino acid sequences of arecombinant SLEV virus E-glycoprotein antigen incorporating the G₁₀₆Qsubstitution.

SEQ ID NOs: 84 and 85 show the nucleic and amino acid sequences of arecombinant WNV virus E-glycoprotein antigen.

SEQ ID NOs: 86 and 87 show the nucleic and amino acid sequences of arecombinant WNV virus E-glycoprotein antigen incorporating the G₁₀₆Vsubstitution.

DETAILED DESCRIPTION I. Abbreviations

ADE antibody-dependant enhancement

Ag-ELISA antigen-capture ELISA

D domain

DEN dengue

DENV dengue virus

E envelope

ELISA enzyme-linked immunoabsorbent assay

IFA indirect immuno-fluorescence assay

JE Japanese encephalitis

JEV Japanese encephalitis virus

mAb monoclonal antibody

MHIAF murine hyper-immune ascetic fluid

MVEV Murray Valley encephalitis virus

PCR polymerase chain reaction

prM premembrane

SLE St. Louis encephalitis

SLEV St. Louis encephalitis virus

TBE tick-borne encephalitis

VLP virus-like particle

WN West Nile

WNV West Nile virus

YF yellow fever

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopediaof Molecular Biology, published by Blackwell Publishers, 1994 (ISBN0632021829); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by Wiley, John& Sons, Inc., 1995 (ISBN 0471186341); and other similar references.

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise. Also, as used herein, the term “comprises” means“includes.” Hence “comprising A or B” means including A, B, or A and B.It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for description.Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including explanations of terms, will control.The materials, methods, and examples are illustrative only and notintended to be limiting.

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects, for example, humans, non-human primates,dogs, cats, horses, and cows.

Antibody: A protein (or protein complex) that includes one or morepolypeptides substantially encoded by immunoglobulin genes or fragmentsof immunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

The basic immunoglobulin (antibody) structural unit is generally atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” (about 50-70 kDa) chain. The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light chain”(V_(L)) and “variable heavy chain” (V_(H)) refer, respectively, to theselight and heavy chains.

As used herein, the term “antibody” includes intact immunoglobulins aswell as a number of well-characterized fragments. For instance, Fabs,Fvs, and single-chain Fvs (SCFvs) that bind to target protein (orepitope within a protein or fusion protein) would also be specificbinding agents for that protein (or epitope). These antibody fragmentsare as follows: (1) Fab, the fragment which contains a monovalentantigen-binding fragment of an antibody molecule produced by digestionof whole antibody with the enzyme papain to yield an intact light chainand a portion of one heavy chain; (2) Fab′, the fragment of an antibodymolecule obtained by treating whole antibody with pepsin, followed byreduction, to yield an intact light chain and a portion of the heavychain; two Fab′ fragments are obtained per antibody molecule; (3)(Fab′)₂, the fragment of the antibody obtained by treating wholeantibody with the enzyme pepsin without subsequent reduction; (4)F(ab′)₂, a dimer of two Fab′ fragments held together by two disulfidebonds; (5) Fv, a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; and (6) single chain antibody, a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule. Methods of makingthese fragments are routine (see, for example, Harlow and Lane, UsingAntibodies: A Laboratory Manual, CSHL, New York, 1999).

Antibodies for use in the methods and compositions of this disclosurecan be monoclonal or polyclonal. Merely by way of example, monoclonalantibodies can be prepared from murine hybridomas according to theclassical method of Kohler and Milstein (Nature 256:495-97, 1975) orderivative methods thereof. Detailed procedures for monoclonal antibodyproduction are described in Harlow and Lane, Using Antibodies: ALaboratory Manual, CSHL, New York, 1999.

Antibody binding affinity: The strength of binding between a singleantibody binding site and a ligand (e.g., an antigen or epitope). Theaffinity of an antibody binding site X for a ligand Y is represented bythe dissociation constant (K_(d)), which is the concentration of Y thatis required to occupy half of the binding sites of X present in asolution. A smaller (K_(d)) indicates a stronger or higher-affinityinteraction between X and Y and a lower concentration of ligand isneeded to occupy the sites. In general, antibody binding affinity can beaffected by the alteration, modification and/or substitution of one ormore amino acids in the epitope recognized by the antibody paratope.

In one example, antibody binding affinity is measured by end-pointtitration in an Ag-ELISA assay. Antibody binding affinity issubstantially lowered (or measurably reduced) by the modification and/orsubstitution of one or more amino acids in the epitope recognized by theantibody paratope if the end-point titer of a specific antibody for themodified/substituted epitope differs by at least 4-fold, such as atleast 10-fold, at least 100-fold or greater, as compared to theunaltered epitope.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T-cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. In one embodiment,an antigen is a flavivirus antigen.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences that determinetranscription. cDNA is synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

Epitope: An antigenic determinant. These are particular chemical groups,such as contiguous or non-contiguous peptide sequences, on a moleculethat are antigenic, that is, that elicit a specific immune response. Anantibody binds a particular antigenic epitope based on the threedimensional structure of the antibody and the matching (or cognate)three dimensional structure of the epitope.

A “cross-reactive epitope” is an epitope found in two or more antigensexpressed by different genes, and responsible for inducingcross-reactive antibodies. For example, a “flavivirus cross-reactiveepitope” is a flavivirus epitope found in a peptide from two or moreflaviviruses, and responsible for inducing flavivirus cross-reactiveantibodies.

A “substituted epitope” comprises at least one structural substitutionin the epitope, such as a substitution of one amino acid for another. Incertain provided embodiments, amino acid substitutions at probable oridentified cross-reactive epitope residues are designed to reduce orablate cross-reactive antibody recognition without substantiallyaltering E-glycoprotein structural conformation or affectingtype-specific antibody binding sites, disrupting dimer interactions, orimpairing particle formation, maturation, or secretion.

Flavivirus cross-reactive antibody: An antibody that recognizes (thatis, specifically binds to) an epitope found on a peptide from more thanone species of flavivirus. Flavivirus cross-reactive antibodies areclassified as either complex cross-reactive or group cross-reactiveantibodies. Complex cross-reactive antibodies recognize epitopes sharedby all viruses within a complex, such as the JE virus complex or the DENvirus complex. Group cross-reactive antibodies recognize epitopes sharedby all members of the genus Flavivirus.

Antibody cross-reactivity is further refined within the sub-complex andsub-group cross-reactive categories. Sub-complex cross-reactiveantibodies recognize epitopes shared by most, but not all, members of aparticular flavivirus complex (e.g., DENV-1, -2, and -3, but notDENV-4), while sub-group cross-reactive antibodies recognize epitopesshared by flaviviruses from several complexes, but not all members ofthe flavivirus group (e.g., all members of the DEN virus and JE viruscomplexes, but not all members of the tick-borne virus complex).Specific, non-limiting examples of flavivirus cross-reactive antibodiesinclude the group cross-reactive mAbs 4G2 and 6B6C-1, the sub-groupcross-reactive mAb 1B7-5, and the sub-complex cross-reactive mAb10A1D-2.

Flavivirus E-glycoprotein: A structural envelope protein that mediatesbinding of flavivirus virions to cellular receptors on host cells. Theflavivirus E-glycoprotein is required for membrane fusion, and is theprimary antigen inducing protective immunity to flavivirus infection.Flavivirus E-glycoprotein affects host range, tissue tropism and viralvirulence. The flavivirus E-glycoprotein contains three structural andfunctional domains, DI-DIII. In mature virus particles theE-glycoprotein forms head to tail homodimers lying flat and forming adense lattice on the viral surface.

Flavivirus E-glycoprotein domain: A domain of a protein is a part of aprotein that shares common structural, physiochemical and/or functionalfeatures; for example hydrophobic, polar, globular, helical domains orproperties, for example a DNA binding domain, an ATP binding domain, andthe like. The flavivirus E-glycoprotein contains three recognizedstructural and functional domains, DI-DIII. DI is an 8-stranded β-barrelcontaining two large insertion loops that form the elongated finger-likeDII. DII is involved in stabilizing the E-glycoprotein dimer andcontains the internal fusion peptide that mediates flaviviral entry intohost cells via membrane fusion. DIII forms a ten-stranded β-barrel withan immunoglobulin-like fold and contains the cellular receptor-bindingmotifs. DI and DIII contain predominately type- and subtype-specificepitopes, whereas DII contains the major flavivirus group and subgroupcross-reactive epitopes, which are sensitive to reduction anddenaturation and are therefore believed to be formed from discontinuousamino acid sequences.

Flavivirus type-specific antibody: An antibody that recognizes (that is,specifically binds to) an epitope found on a peptide from only onespecific member of the flaviviruses. Specific, non-limiting examples offlavivirus type-specific antibodies include: DI mAb 9A4D-1, DII mAb1A5D-1, and DIII mAbs 3H5, 9A3D-8 and 9D12, which only recognizeepitopes found in the DENV-2 E-glycoprotein.

Hybridization: Oligonucleotides and their analogs hybridize by hydrogenbonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary bases. Generally, nucleic acidconsists of nitrogenous bases that are either pyrimidines (cytosine (C),uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)).These nitrogenous bases form hydrogen bonds between a pyrimidine and apurine, and the bonding of the pyrimidine to the purine is referred toas “base pairing.” More specifically, A will hydrogen bond to T or U,and G will bond to C. “Complementary” refers to the base pairing thatoccurs between to distinct nucleic acid sequences or two distinctregions of the same nucleic acid sequence.

“Specifically hybridizable” and “specifically complementary” are termsthat indicate a sufficient degree of complementarity such that stableand specific binding occurs between the oligonucleotide (or its analog)and the DNA or RNA target. The oligonucleotide or oligonucleotide analogneed not be 100% complementary to its target sequence to be specificallyhybridizable. An oligonucleotide or analog is specifically hybridizablewhen binding of the oligonucleotide or analog to the target DNA or RNAmolecule interferes with the normal function of the target DNA or RNA,and there is a sufficient degree of complementarity to avoidnon-specific binding of the oligonucleotide or analog to non-targetsequences under conditions where specific binding is desired, forexample under physiological conditions in the case of in vivo assays orsystems. Such binding is referred to as specific hybridization.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing nucleic acidsequences. Generally, the temperature of hybridization and the ionicstrength (especially the Na⁺ and/or Mg⁺⁺ concentration) of thehybridization buffer will determine the stringency of hybridization,though wash times also influence stringency. Calculations regardinghybridization conditions required for attaining particular degrees ofstringency are discussed by Sambrook et al. (ed.), Molecular Cloning: ALaboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989, chapters 9 and 11; and Ausubel etal. Short Protocols in Molecular Biology, 4^(th) ed., John Wiley & Sons,Inc., 1999.

For purposes of the present disclosure, “stringent conditions” encompassconditions under which hybridization will only occur if there is lessthan 25% mismatch between the hybridization molecule and the targetsequence. “Stringent conditions” may be broken down into particularlevels of stringency for more precise definition. Thus, as used herein,“moderate stringency” conditions are those under which molecules withmore than 25% sequence mismatch will not hybridize; conditions of“medium stringency” are those under which molecules with more than 15%mismatch will not hybridize, and conditions of “high stringency” arethose under which sequences with more than 10% mismatch will nothybridize. Conditions of “very high stringency” are those under whichsequences with more than 6% mismatch will not hybridize.

“Specific hybridization” refers to the binding, duplexing, orhybridizing of a molecule only or substantially only to a particularnucleotide sequence when that sequence is present in a complex mixture(for example, total cellular DNA or RNA). Specific hybridization mayalso occur under conditions of varying stringency.

Immune stimulatory composition: A term used herein to mean a compositionuseful for stimulating or eliciting a specific immune response (orimmunogenic response) in a vertebrate. The immune stimulatorycomposition can be a protein antigen or a plasmid vector used to expressa protein antigen. In some embodiments, the immunogenic response isprotective or provides protective immunity, in that it enables thevertebrate animal to better resist infection with or disease progressionfrom the organism against which the immune stimulatory composition isdirected.

Without wishing to be bound by a specific theory, it is believed that animmunogenic response induced by an immune stimulatory composition mayarise from the generation of an antibody specific to one or more of theepitopes provided in the immune stimulatory composition. Alternatively,the response may comprise a T-helper or cytotoxic cell-based response toone or more of the epitopes provided in the immune stimulatorycomposition. All three of these responses may originate from naïve ormemory cells. One specific example of a type of immune stimulatorycomposition is a vaccine.

In some embodiments, an “effective amount” or “immune-stimulatoryamount” of an immune stimulatory composition is an amount which, whenadministered to a subject, is sufficient to engender a detectable immuneresponse. Such a response may comprise, for instance, generation of anantibody specific to one or more of the epitopes provided in the immunestimulatory composition. Alternatively, the response may comprise aT-helper or CTL-based response to one or more of the epitopes providedin the immune stimulatory composition. All three of these responses mayoriginate from naïve or memory cells. In other embodiments, a“protective effective amount” of an immune stimulatory composition is anamount which, when administered to a subject, is sufficient to conferprotective immunity upon the subject.

Inhibiting or treating a disease: Inhibiting the full development of adisease or condition, for example, in a subject who is at risk for adisease. Specific examples of diseases include dengue fever, denguehemorrhagic fever, yellow fever, Japanese encephalitis, tick-borneencephalitis, and West Nile disease. “Treatment” refers to a therapeuticintervention that ameliorates a sign or symptom of a disease orpathological condition after it has begun to develop. As used herein,the term “ameliorating,” with reference to a disease, pathologicalcondition or symptom, refers to any observable beneficial effect of thetreatment. The beneficial effect can be evidenced, for example, by adelayed onset of clinical symptoms of the disease in a susceptiblesubject, a reduction in severity of some or all clinical symptoms of thedisease, a slower progression of the disease, a reduction in the numberof relapses of the disease, an improvement in the overall health orwell-being of the subject, or by other parameters well known in the artthat are specific to the particular disease.

Isolated: An “isolated” or “purified” biological component (such as anucleic acid, peptide, protein, protein complex, or particle) has beensubstantially separated, produced apart from, or purified away fromother biological components in the cell of the organism in which thecomponent naturally occurs, that is, other chromosomal andextrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides andproteins that have been “isolated” or “purified” thus include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids, peptides and proteins prepared byrecombinant expression in a host cell, as well as chemically synthesizednucleic acids or proteins. The term “isolated” or “purified” does notrequire absolute purity; rather, it is intended as a relative term.Thus, for example, an isolated biological component is one in which thebiological component is more enriched than the biological component isin its natural environment within a cell, or other production vessel.Preferably, a preparation is purified such that the biological componentrepresents at least 50%, such as at least 70%, at least 90%, at least95%, or greater, of the total biological component content of thepreparation.

Nucleic acid molecule: A polymeric form of nucleotides, which mayinclude both sense and anti-sense strands of RNA, cDNA, genomic DNA, andsynthetic forms and mixed polymers of the above. A nucleotide refers toa ribonucleotide, deoxynucleotide or a modified form of either type ofnucleotide. The term “nucleic acid molecule” as used herein issynonymous with “nucleic acid” and “polynucleotide.” A nucleic acidmolecule is usually at least 10 bases in length, unless otherwisespecified. The term includes single- and double-stranded forms of DNA. Apolynucleotide may include either or both naturally occurring andmodified nucleotides linked together by naturally occurring and/ornon-naturally occurring nucleotide linkages.

Oligonucleotide: A nucleic acid molecule generally comprising a lengthof 300 bases or fewer. The term often refers to single-strandeddeoxyribonucleotides, but it can refer as well to single- ordouble-stranded ribonucleotides, RNA:DNA hybrids and double-strandedDNAs, among others. The term “oligonucleotide” also includesoligonucleosides (that is, an oligonucleotide minus the phosphate) andany other organic base polymer.

In some examples, oligonucleotides are about 10 to about 90 bases inlength, for example, 12, 13, 14, 15, 16, 17, 18, 19 or 20 bases inlength. Other oligonucleotides are about 25, about 30, about 35, about40, about 45, about 50, about 55, about 60 bases, about 65 bases, about70 bases, about 75 bases or about 80 bases in length. Oligonucleotidesmay be single-stranded, for example, for use as probes or primers, ormay be double-stranded, for example, for use in the construction of amutant gene. Oligonucleotides can be either sense or anti-senseoligonucleotides. An oligonucleotide can be modified as discussed abovein reference to nucleic acid molecules. Oligonucleotides can be obtainedfrom existing nucleic acid sources (for example, genomic or cDNA), butcan also be synthetic (for example, produced by laboratory or in vitrooligonucleotide synthesis).

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence is the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, in the samereading frame. If introns are present, the operably linked DNA sequencesmay not be contiguous.

Paratope: That portion of an antibody that is responsible for itsbinding to an antigenic determinant (epitope) on an antigen.

Polypeptide: A polymer in which the monomers are amino acid residuesjoined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used, the L-isomers being preferred for many biological uses. Theterms “polypeptide” or “protein” as used herein are intended toencompass any amino acid molecule and include modified amino acidmolecules such as glycoproteins. The term “polypeptide” is specificallyintended to cover naturally occurring proteins, as well as those whichare recombinantly or synthetically produced.

Probes and primers: A probe comprises an isolated nucleic acid moleculeattached to a detectable label or other reporter molecule. Typicallabels include radioactive isotopes, enzyme substrates, co-factors,ligands, chemiluminescent or fluorescent agents, haptens, and enzymes.Methods for labeling and guidance in the choice of labels appropriatefor various purposes are discussed, for example, in Sambrook et al.(ed.), Molecular Cloning: A Laboratory Manual, 2^(nd) ed., vol. 1-3,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 andAusubel et al. Short Protocols in Molecular Biology, 4^(th) ed., JohnWiley & Sons, Inc., 1999.

Primers are short nucleic acid molecules, for instance DNAoligonucleotides 6 nucleotides or more in length, for example thathybridize to contiguous complementary nucleotides or a sequence to beamplified. Longer DNA oligonucleotides may be about 10, 12, 15, 20, 25,30, or 50 nucleotides or more in length. Primers can be annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, and then the primerextended along the target DNA strand by a DNA polymerase enzyme. Primerpairs can be used for amplification of a nucleic acid sequence, forexample, by the polymerase chain reaction (PCR) or other nucleic-acidamplification methods known in the art. Other examples of amplificationinclude strand displacement amplification, as disclosed in U.S. Pat. No.5,744,311; transcription-free isothermal amplification, as disclosed inU.S. Pat. No. 6,033,881; repair chain reaction amplification, asdisclosed in WO 90/01069; ligase chain reaction amplification, asdisclosed in EP-A-320 308; gap filling ligase chain reactionamplification, as disclosed in U.S. Pat. No. 5,427,930; and NASBA™ RNAtranscription-free amplification, as disclosed in U.S. Pat. No.6,025,134.

Methods for preparing and using nucleic acid probes and primers aredescribed, for example, in Sambrook et al. (ed.), Molecular Cloning: ALaboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989; Ausubel et al. Short Protocols inMolecular Biology, 4^(th) ed., John Wiley & Sons, Inc., 1999; and Inniset al. PCR Protocols, A Guide to Methods and Applications, AcademicPress, Inc., San Diego, Calif., 1990. Amplification primer pairs can bederived from a known sequence, for example, by using computer programsintended for that purpose such as Primer (Version 0.5., © 1991,Whitehead Institute for Biomedical Research, Cambridge, Mass.). One ofordinary skill in the art will appreciate that the specificity of aparticular probe or primer increases with its length. Thus, in order toobtain greater specificity, probes and primers can be selected thatcomprise at least 20, 25, 30, 35, 40, 45, 50 or more consecutivenucleotides of a target nucleotide sequences.

Recombinant nucleic acid: A nucleic acid molecule that is not naturallyoccurring or has a sequence that is made by an artificial combination oftwo otherwise separated segments of sequence. This artificialcombination is accomplished by chemical synthesis or, more commonly, bythe artificial manipulation of isolated segments of nucleic acids, e.g.,by genetic engineering techniques such as those described in Sambrook etal. (ed.), Molecular Cloning: A Laboratory Manual, 2^(nd) ed., vol. 1-3,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Theterm recombinant includes nucleic acids that have been altered solely byaddition, substitution, or deletion of a portion of a natural nucleicacid molecule.

Regulatory sequences or elements: These terms refer generally to a classof DNA sequences that influence or control expression of genes. Includedin the term are promoters, enhancers, locus control regions (LCRs),insulators/boundary elements, silencers, matrix attachment regions(MARS, also referred to as scaffold attachment regions), repressor,transcriptional terminators, origins of replication, centromeres, andmeiotic recombination hotspots. Promoters are sequences of DNA near the5′-end of a gene that act as a binding site for DNA-dependent RNApolymerase, and from which transcription is initiated. Enhancers arecontrol elements that elevate the level of transcription from apromoter, usually independently of the enhancer's orientation ordistance from the promoter. LCRs confer tissue-specific and temporallyregulated expression to genes to which they are linked. LCRs functionindependently of their position in relation to the gene, but arecopy-number dependent. It is believed that they function to open thenucleosome structure, so other factors can bind to the DNA. LCRs mayalso affect replication timing and origin usage. Insulators (also knowas boundary elements) are DNA sequences that prevent the activation (orinactivation) of transcription of a gene, by blocking effects ofsurrounding chromatin. Silencers and repressors are control elementsthat suppress gene expression; they act on a gene independently of theirorientation or distance from the gene. MARs are sequences within DNAthat bind to the nuclear scaffold; they can affect transcription,possibly by separating chromosomes into regulatory domains. It isbelieved that MARs mediate higher-order, looped structures withinchromosomes. Transcriptional terminators are regions within the genevicinity where RNA Polymerase is released from the template. Origins ofreplication are regions of the genome, during DNA synthesis orreplication phases of cell division, that begin the replication processof DNA. Meiotic recombination hotspots are regions of the genome thatrecombine more frequently than average during meiosis.

Sequence identity: The similarity between two nucleic acid sequences, ortwo amino acid sequences, is expressed in terms of the similaritybetween the sequences, otherwise referred to as sequence identity.Sequence identity is frequently measured in terms of percentage identity(or similarity or homology); the higher the percentage, the more similarthe two sequences are.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman (Adv. Appl. Math., 2:482, 1981); Needleman and Wunsch (J.Mol. Biol., 48:443, 1970); Pearson and Lipman (Proc. Natl. Acad. Sci.,85:2444, 1988); Higgins and Sharp (Gene, 73:237-44, 1988); Higgins andSharp (CABIOS, 5:151-53, 1989); Corpet et al. (Nuc. Acids Res.,16:10881-90, 1988); Huang et al. (Comp. Appls. Biosci., 8:155-65, 1992);and Pearson et al. (Meth. Mol. Biol., 24:307-31, 1994). Altschul et al.(Nature Genet., 6:119-29, 1994) presents a detailed consideration ofsequence alignment methods and homology calculations.

The alignment tools ALIGN (Myers and Miller, CABIOS 4:11-17, 1989) orLFASTA (Pearson and Lipman, 1988) may be used to perform sequencecomparisons (Internet Program © 1996, W. R. Pearson and the Universityof Virginia, “fasta20u63” version 2.0u63, release date December 1996).ALIGN compares entire sequences against one another, while LFASTAcompares regions of local similarity. These alignment tools and theirrespective tutorials are available on the Internet at the NCSA website.Alternatively, for comparisons of amino acid sequences of greater thanabout 30 amino acids, the “Blast 2 sequences” function can be employedusing the default BLOSUM62 matrix set to default parameters, (gapexistence cost of 11, and a per residue gap cost of 1). When aligningshort peptides (fewer than around 30 amino acids), the alignment shouldbe performed using the “Blast 2 sequences” function, employing the PAM30matrix set to default parameters (open gap 9, extension gap 1penalties). The BLAST sequence comparison system is available, forinstance, from the NCBI web site; see also Altschul et al., J. Mol.Biol., 215:403-10, 1990; Gish. and States, Nature Genet., 3:266-72,1993; Madden et al., Meth. Enzymol., 266:131-41, 1996; Altschul et al.,Nucleic Acids Res., 25:3389-402, 1997; and Zhang and Madden, GenomeRes., 7:649-56, 1997.

Orthologs (equivalent to proteins of other species) of proteins are insome instances characterized by possession of greater than 75% sequenceidentity counted over the full-length alignment with the amino acidsequence of specific protein using ALIGN set to default parameters.Proteins with even greater similarity to a reference sequence will showincreasing percentage identities when assessed by this method, such asat least 80%, at least 85%, at least 90%, at least 92%, at least 95%, orat least 98% sequence identity. In addition, sequence identity can becompared over the full length of one or both binding domains of thedisclosed fusion proteins.

When significantly less than the entire sequence is being compared forsequence identity, homologous sequences will typically possess at least80% sequence identity over short windows of 10-20, and may possesssequence identities of at least 85%, at least 90%, at least 95%, or atleast 99% depending on their similarity to the reference sequence.Sequence identity over such short windows can be determined usingLFASTA; methods are described at the NCSA website. One of skill in theart will appreciate that these sequence identity ranges are provided forguidance only; it is entirely possible that strongly significanthomologs could be obtained that fall outside of the ranges provided.Similar homology concepts apply for nucleic acids as are described forprotein. An alternative indication that two nucleic acid molecules areclosely related is that the two molecules hybridize to each other understringent conditions.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences, due to the degeneracyof the genetic code. It is understood that changes in nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid sequences that each encode substantially the same protein.

Specific binding agent: An agent that binds substantially only to adefined target. Thus a protein-specific binding agent bindssubstantially only the defined protein, or to a specific region withinthe protein. As used herein, protein-specific binding agents includeantibodies and other agents that bind substantially to a specifiedpolypeptide. The antibodies may be monoclonal or polyclonal antibodiesthat are specific for the polypeptide, as well as immunologicallyeffective portions (“fragments”) thereof.

The determination that a particular agent binds substantially only to aspecific polypeptide may readily be made by using or adapting routineprocedures. Examples of suitable in vitro assays which make use of theWestern blotting procedure include IFA and Ag-ELISA, and are describedin many standard texts, including Harlow and Lane, Using Antibodies: ALaboratory Manual, CSHL, New York, 1999.

Transformed: A “transformed” cell is a cell into which has beenintroduced a nucleic acid molecule by molecular biology techniques. Theterm encompasses all techniques by which a nucleic acid molecule mightbe introduced into such a cell, including transfection with viralvectors, transformation with plasmid vectors, and introduction of nakedDNA by electroporation, lipofection, and particle gun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergenes and other genetic elements known in the art.

III. Overview of Several Embodiments

Isolated mutant flavivirus polypeptides exhibiting measurably reducedantibody cross-reactivity (compared to corresponding wild-typepolypeptides) are disclosed herein. In one embodiment, the isolatedflavivirus polypeptides are flavivirus E-glycoproteins that include anamino acid sequence as shown in SEQ ID NO: 14, wherein at least one ofthe amino acids at position 104, 106, 107, 126, 226, or 231 issubstituted (compared to corresponding wild-type E-glycoproteins).Specific, non-limiting examples of the amino acid substitutions atpositions 104, 106, 107, 126, 226, and 231 include: G₁₀₄H (SEQ ID NO:16), G₁₀₆Q (SEQ ID NO: 18), L₁₀₇K (SEQ ID NO: 20), E₁₂₆A (SEQ ID NO:22), T₂₂₆N (SEQ ID NO: 24), W₂₃₁F (SEQ ID NO: 26), and W₂₃₁L (SEQ ID NO:28). Also disclosed are isolated nucleic acid molecules encoding theflavivirus polypeptides with at least one amino acid substitution atposition 104, 106, 107, 126, 226, or 231 of SEQ ID NO: 14.Representative nucleic acid molecules are shown in SEQ ID NOs: 15, 17,19, 21, 23, 25, and 27.

In another embodiment, the isolated flavivirus polypeptides areflavivirus E-glycoproteins that include an amino acid sequence as shownin SEQ ID NO: 81, wherein at least one of the amino acids at position106 is substituted (compared to corresponding wild-typeE-glycoproteins). Specific, non-limiting examples of the amino acidsubstitutions at position 106 include: G₁₀₆Q (SEQ ID NO: 83). Alsodisclosed are isolated nucleic acid molecules encoding the flaviviruspolypeptides with at least one amino acid substitution at position 106of SEQ ID NO: 81. A representative nucleic acid molecule is shown in SEQID NO: 82.

In yet another embodiment, the isolated flavivirus polypeptides areflavivirus E-glycoproteins that include an amino acid sequence as shownin SEQ ID NO: 85, wherein at least one of the amino acids at position106 is substituted (compared to corresponding wild-typeE-glycoproteins). Specific, non-limiting examples of the amino acidsubstitutions at position 106 include: G₁₀₆V (SEQ ID NO: 87). Alsodisclosed are isolated nucleic acid molecules encoding the flaviviruspolypeptides with at least one amino acid substitution at position 106of SEQ ID NO: 85. A representative nucleic acid molecule is shown in SEQID NO: 86.

Pharmaceutical and immune stimulatory compositions are also disclosedthat include one or more flavivirus E-glycoprotein polypeptidesexhibiting measurably reduced antibody cross-reactivity, with at leastone amino acid substitution at position 104, 106, 107, 126, 226, or 231of SEQ ID NO: 14. Also disclosed are pharmaceutical and immunestimulatory compositions that include one or more nucleic acid moleculesencoding the flavivirus polypeptides with at least one amino acidsubstitution at position 104, 106, 107, 126, 226, or 231 of SEQ ID NO:14. Representative nucleic acid molecules are shown in SEQ ID NOs: 15,17, 19, 21, 23, 25, and 27.

Also disclosed are pharmaceutical and immune stimulatory compositionsthat include one or more flavivirus E-glycoprotein polypeptidesexhibiting measurably reduced antibody cross-reactivity, with at leastone amino acid substitution at position 106 of SEQ ID NO: 81 or SEQ IDNO: 85. Also disclosed are pharmaceutical and immune stimulatorycompositions that include one or more nucleic acid molecules encodingthe flavivirus polypeptides with at least one amino acid substitution atposition 106 of SEQ ID NO: 81 or SEQ ID NO: 85. Representative nucleicacid molecules are shown in SEQ ID NOs: 82 and 86.

In another embodiment, a method is provided for identifying andmodifying a flavivirus cross-reactive epitope. This method includesselecting a candidate cross-reactive epitope using a structure-baseddesign approach, and designing a substituted epitope including at leastone amino acid residue substitution compared to the candidate epitope.The candidate epitope is then contacted with a specific binding agentunder conditions whereby a candidate epitope/specific binding agentcomplex can form. Likewise, the substituted epitope is contacted withthe same specific binding agent under the same conditions used forcandidate epitope/specific binding agent complex formation. A candidateepitope is identified as a flavivirus cross-reactive epitope when thesubstituted epitope has a substantially lower binding affinity for thespecific binding agent compared to the candidate epitope, and whereinthe flavivirus cross-reactive epitope binds to a specific binding agentthat binds to at least two flaviviruses. In specific, non-limitingexamples, the at least two flaviviruses are selected from dengueserotype 1 virus, dengue serotype 2 virus, dengue serotype 3 virus,dengue serotype 4 virus, yellow fever virus, Japanese encephalitisvirus, St. Louis encephalitis virus, and West Nile virus. In yet anotherspecific example of the provided method, the specific binding agent is aflavivirus cross-reactive antibody.

In a further specific example of the provided method, thestructure-based design approach includes identifying at least oneconserved flavivirus amino acid between two or more flavivirus groups orsubgroups, and mapping the conserved flavivirus amino acid onto astructure of a flavivirus E-glycoprotein. In another specific example,the conserved flavivirus amino acid exhibits two or more of thefollowing structural characteristics: it is located in DII of theE-glycoprotein, it is conserved across the flaviviruses, it is on theouter or lateral surface of the E-glycoprotein dimer, it has at least35% surface accessibility potential, its side chain projection isaccessible for antibody paratopes, or it has a high β-factor.

In yet a further specific example of the provided method, thestructure-based design approach includes identifying at least oneconserved flavivirus amino acid between two or more flavivirus complexesor subcomplexes, and mapping the conserved flavivirus amino acid onto astructure of a flavivirus E-glycoprotein. In still another specificexample, the conserved flavivirus amino acid exhibits two or more of thefollowing structural characteristics: it has at least 35% surfaceaccessibility potential, it is on the outer or lateral surface of theE-glycoprotein dimer, it is conserved across the flaviviruses, its sidechain projection is accessible for antibody paratopes, or it has a highβ-factor.

In another embodiment, a method is provided for detecting a flavivirusantibody in a sample. This method includes contacting the sample withthe disclosed mutant flavivirus polypeptides under conditions whereby apolypeptide/antibody complex can form, and detectingpolypeptide/antibody complex formation, thereby detecting a flavivirusantibody in a sample. Also disclosed are methods of diagnosing aflavivirus infection in a subject. In one embodiment, the methodincludes contacting a sample from the subject with the disclosed mutantflavivirus polypeptides under conditions whereby a polypeptide/antibodycomplex can form, and detecting polypeptide/antibody complex formation,thereby diagnosing a flavivirus infection in a subject.

Also disclosed is a flavivirus E-glycoprotein engineered to comprise atleast one amino acid residue substitution according to the methodsdescribed herein.

IV. Identifying Flavivirus Cross-Reactive Epitopes

The current disclosure provides methods for identifying flaviviruscross-reactive epitopes, as well as distinguishing such epitopes fromspecies-specific (or type-specific) epitopes.

In one embodiment, the method comprises a structure-based designapproach, which optionally includes one or more of the followingrequirements in order to identify cross-reactive epitopes: 1) theepitope is located in DII of the E-glycoprotein, for example, aminoacids 52-135 and 195-285 in the TBE virus E-glycoprotein, 52-132 and193-280 in the DEN-2 virus E-glycoprotein, and conserved across theflaviviruses or multiple flaviviral species; 2) the epitope is on theouter or lateral surface of the E-glycoprotein dimer; 3) the epitope hasat least 35% surface accessibility potential; 4) one or more side chainprojections of amino acids within the epitope are accessible to antibodyparatopes; and 5) residues with high temperature (β) factors arefavored.

In one embodiment, a structure-based design approach comprises aprocedural algorithm developed to localize epitopes responsible forinducing flavivirus cross-reactive antibodies. Strictly-conservedflavivirus residues are initially identified. These residues are mapped,for example, onto a 2.0 Å resolution E-glycoprotein structure for TBEvirus (Rey et al., Nature 375:291-98, 1995), a high resolution DEN-2virus E-glycoprotein structure (Modis et al., PNAS 100:6986-91, 2003),or other similar structure. Optionally, strictly-conserved flavivirusresidues are also mapped onto a computer predicted homology modelstructure for the DEN-2 virus E-glycoprotein using, for example, theSwiss-Pdb Viewer 3.7 structure analysis software (Guex et al.,Electrophoresis 18:2714-23, 1997).

The following criteria (individually or in combination of two or more)are then employed in certain embodiments to select probable flavivirusgroup or subgroup cross-reactive epitope residues: 1) an amino acidlocated in DII (for example, amino acids 52-135 and 195-285 in the TBEvirus E-glycoprotein (Rey et al., Nature 375:291-98, 1995); 52-132 and193-280 in the DEN-2 virus E-glycoprotein (Modis et al., PNAS100:6986-91, 2003)), and conserved among more than one flavivirus; 2)amino acids on the outer or lateral surface of the E-glycoprotein dimer;3) amino acids with at least 35% surface accessibility potential; 4)side chain projections accessible to antibody paratopes; and 5) residueswith high temperature (β) factors should be favored, as these residuestend to be flexible and are able to conform to the antibody paratope,increasing the antibody-antigen (Ab-Ag) affinity.

Similar criteria (individually or in combination of two or more) areemployed in certain embodiments to select probable flavivirus complex orsubcomplex cross-reactive epitope residues. The procedural algorithm forthe identification of flavivirus complex and sub-complex cross-reactiveepitopes utilizes the following optimality criteria: 1) Theidentification and selection of amino acid residues with ≧35% of theirsurface solvent accessible. These residues are identified from thepublished atomic structure coordinates of the DENV-2 soluble ectodomainof the envelope glycoprotein and homology models of SLEV and WNV derivedfrom the DENV-2 structure (Modis et al., Proc. Natl. Acad. Sci. USA100:6986-91, 2003). In addition to examination of amino acid residues instructural domain II, residues in domains I and III were examined, sincepublished results indicate that some complex and sub-complexcross-reactive epitopes are mapped onto domains I and III in addition todomain II (Roehrig et al., Virology 246:317-28, 1998). 2) Amino acids onthe outer or lateral surface of the E-glycoprotein dimer, and accessibleto antibody. 3) Amino acid conservation across the flavivirus complex(based upon a structural alignment of the protein sequences). Residuesconserved across all member viruses of the same complex are favored. Ifconserved within but not across the entire complex, then residues withshared identities between WNV and SLEV are favored in the JEV complex,and residues with shared identities between DENV-2 and two or more otherviruses in the DENV complex are favored over those shared with DENV-2and only one other DENV complex virus. 4) Side chain projections exposedtowards the outer surface and accessible to antibody paratopes. 5)Residues with high temperature (β-) factors should be favored, as theseresidues tend to be flexible and are able to conform to the antibodyparatope, increasing the antibody-antigen affinity. Amino acid residueswith high temperature factors are more commonly found in antigenepitopes than lower temperature factor residues. 6) Followingidentification of potential individual flavivirus complex andsub-complex cross-reactive epitope residues, all residues are mapped andhighlighted on the same E-glycoprotein dimer structure together. Withthis technique, groups of potential cross-reactive epitope residuesforming clusters (and hence probable epitopes) are readily identified.7) Residues fitting all of these criteria and occurring in structuralclusters approximately 20×30 Å² (which is the average “footprint” of anantibody Fab that interacts with an antigen epitope) are favored overresidues that are more isolated in the protein structure. 8) Within anidentified structural cluster of potential epitope residues, residuesthat more completely satisfy greater numbers of the optimality criteriaare selected for the first round of mutagenesis analysis.

A. Outer and/or Lateral Surface Amino Acids

In one embodiment, the outer and/or lateral surface of theE-glycoprotein dimer comprises those residues which are exposed on thesurface of the E-glycoprotein dimer in a way that they are physicallycapable of interacting with a host-derived immunoglobulin antibodymolecule. The flavivirus virion contains a host cell-derived lipidbilayer, with E-glycoprotein dimers imbedded within this lipid bilayervia their trans-membrane domains. The ectodomains of the E-glycoproteindimers lie on top of this bilayer, forming a dense lattice andessentially coating the virion in a protein shell. Because of thisstructural organization, there are regions of the E-glycoprotein that,under general assembled virion conditions, cannot physically interactwith an immunoglobulin molecule, and therefore are highly unlikely toform part of an antibody epitope. Such inaccessible regions include thetrans-membrane domains (because they are imbedded within the lipidbilayer and are covered by the ectodomain) and more than two-thirds ofthe residues of the ectodomain itself, which are either on the bottomsurface of the dimer (and therefore packed between the lipid layer andthe ectodomain), or are packed into the interior of this globularprotein rather than on its surface. Because of these structuralconstraints, under normal conditions immunoglobulin molecules can onlyinteract with residues on the outer exposed surface of theE-glycoprotein dimer, and with a subset of residues on the outer lateralsurface. Because of the close packing of E-glycoprotein dimers into anetwork across the surface of the virion, and the difficulty of a largeimmunoglobulin molecule accessing these narrow spaces, it is believedthat only some of the lateral surface residues are available forimmunoglobulin interaction. For these reasons, only residues located onthe outer or lateral surface of the E-glycoprotein are considered asparticipating in possible flavivirus cross-reactive epitopes. Aninspection of the location of a residue (e.g., a residue conserved amongmore than one flavivirus, such as Gly₁₀₄, Gly₁₀₆, Leu₁₀₇, or Trp₂₃₁) inthe E-glycoprotein dimer atomic structure allows for a determination asto whether or not a residue is located on the outer or lateral surfaceof the dimer.

B. Surface Accessibility Potential

In one embodiment, surface accessibility potential comprises thatportion of the predicted electron density surrounding any amino acidresidue's side chain that is exposed on the surface of the protein, andtheoretically available to interact with another molecule. For any given“surface” residue, its surface accessibility is affected by the local(and surrounding) secondary structure of the alpha-carbon main chain,and the positions and types of immediately surrounding side-chainprojections. Thus, by definition, maximum accessibility would be for aresidue X in the peptide GGXGG in an extended conformation, as theglycine residues have no side chains and therefore amino acid X'ssurface accessibility is not constrained by either the alpha-carbonbackbone shape or the surrounding residues' side chain projections (see,e.g., Li et al., Nature Struct. Bio. 10:482-88, 2003; and Faelber etal., J. Mol. Biol. 313:83-97, 2001).

C. Accessible Side Chain Projections

In one embodiment, the side chain projection(s) accessible for antibodyparatopes comprises a qualitative assessment of how exposed and/oravailable a given amino acid's reactive side chain is to interact with ahypothetical immunoglobulin molecule. The angle of projection of a sidechain is determined primarily by its position in the primary amino acidchain. However, upon folding of this polypeptide chain, the side chainprojections are additionally altered or affected by electrostatic andother forces from surrounding residues. The accessibility of an aminoacid's side chain projections to be bound by antibody is a specificcriterion that is inherent in an amino acid's “surface accessibility.”Hence, theoretical amino acid X could have 50% surface accessibility andyet its side-chain may still be directed towards the interior of theprotein and therefore be unlikely to interact energetically with animmunoglobulin molecule (see, e.g., Li et al., Nature Struct. Bio.10:482-88, 2003; Faelber et al., J. Mol. Biol. 313:83-97, 2001; and Eyalet al., J. Comp. Chem. 25: 712-24, 2003).

D. High Temperature Factors

In one embodiment, a temperature or β-factor comprises a criterion whichrepresents a particular amino acid's potential flexibility within theprotein. Any given atom within a protein structure is defined by fourparameters, the three x, y and z coordinates, defining its position inspace, and its β- or temperature factor. For well defined,high-resolution crystal structures, β-values are typically ≦20 Å². Highβ-values, for example, ≧40 Å² can be a signal that there is littleconfidence in the assignment of these atoms within the protein (forexample, if the protein is disordered and does not consistently foldinto the same structure). However, in well-defined atomic-levelresolution protein structures, high β-factors associated with particularatoms for individual amino acids are typically interpreted as indicatorsof that residue or atom's potential flexibility. This criterion isrelevant to epitope determination, as shape complementarity of themolecular surfaces of both the antibody paratope and the antigen epitopeis know to be an important factor effecting antibody avidity. Flexibleresidues, identified by their higher β-factors, are better able to makeslight positional adjustments, thereby improving shape complementarityand the energetics of the Ag-Ab interaction. It has been demonstratedthat epitope amino acids involved in antibody interactions are morelikely to have high β-factors than are amino acids from the same proteinthat do not interact with antibodies (see, e.g., Mylvaganam et al., J.Mol. Biol. 281:301-22, 1998).

Amino acid substitutions at probable cross-reactive epitope residues aremodeled, selecting substitutions that should reduce or ablate antibodyrecognition without altering E-glycoprotein structural conformation,disrupting dimer interactions, or impairing particle formation,maturation, or secretion. For this reason, cysteine residues otherwisesatisfying the cross-reactive epitope criteria are not recommended formutagenesis because their involvement in disulphide bridging is believedto be necessary for proper E-glycoprotein structure and function (Modiset al., PNAS 100:6986-91, 2003; Rey et al., Nature 375:291-98, 1995).Stability calculations are performed for all possible amino acidsubstitutions of candidate residues using, for example, the FOLD-Xserver (Guerois et al., J. Mol. Biol. 320:369-87, 2002; available on theinternet) and the TBE virus E-glycoprotein pdb file coordinates (Rey etal., Nature 375:291-98, 1995). By way of example, amino acidsubstitutions modeled in the TBE virus E-glycoprotein with free energiesof folding equal to or less than that of the non-mutated wild-typeE-glycoprotein are re-examined with the Swiss-PdbViewer software, toidentify those substitutions that minimized local structuraldisturbances while maintaining structurally relevant biochemicalinteractions such as hydrogen bonding and/or charge interactions withneighboring amino acids.

Optionally, upon the successful identification of cross-reactive epitoperesidues, the E-glycoprotein structure can be further analyzed toidentify additional residues forming cross-reactive epitopes. By way ofexample, a “nearest neighbor” search is conducted of the surface of theE-glycoprotein structure, looking for additional residues located within10-15 Å of the identified residue. This distance is within the bindingfootprint of a single antibody paratope (Faebler et al., J. Mol. Biol.313:83-97, 2001). In this second iteration of cross-reactive epitoperesidue identification, the same five optimality criterion as above areused, with one change. The criterion of strict conservation across theflaviviruses is relaxed to now include variable residues. In this way,residues either conserved in their physiochemical nature and/orconserved only within a particular flavivirus complex (such as the fourDEN serotypes) or subgroup can be identified.

Also provided are methods for designing a substituted epitope comprisingat least one amino acid residue substitution compared to a wild-typecandidate epitope; obtaining a first sample comprising the candidateepitope; obtaining a second sample comprising the substituted epitope;contacting the first sample with a specific binding agent; andcontacting the second sample with the specific binding agent, whereinthe cross-reactive epitope is identified when the substituted epitopehas a substantially lower binding affinity for the specific bindingagent compared to the candidate epitope. Antibody binding affinities canbe determined by many methods well known in the art, such as end-pointtitration in an Ag-ELISA assay, competition binding in an ELISA assay, asolid-phase radioimmunoassay, and the Biacore® surface plasmon resonancetechnique (Malmqvist, Biochem. Soc. Trans. 27:335-40, 1999; and Drake etal., Anal. Biochem. 328:35-43, 2004).

In some embodiments the specific binding agent is an antibody, forexample, a polyclonal antibody or a mAb. A specific, non-limitingexample of a polyclonal antibody is polyclonal anti-DEN-2 MHIAF.Specific, non-limiting examples of mAbs include 4G2 (ATCC No. HB-112),6B6C-1, 1B7-5, 10A1D-2, 1A5D-1, and 1B4C-2 (Roehrig et al., Virology246:317-28, 1998).

V. Flavivirus Cross-Reactive Epitopes and Variants Thereof

The disclosure also provides an isolated polypeptide comprising at leastone flavivirus cross-reactive epitope residue, wherein the antibodycross-reactivity of the at least one flavivirus cross-reactive epitopehas been reduced or ablated. In one embodiment, one or more amino acidsubstitutions of one or more flavivirus cross-reactive epitope residuescauses the reduction or ablation of antibody cross-reactivity. Inanother embodiment, the at least one flavivirus cross-reactive epitoperesidue with reduced or ablated cross-reactivity has measurably lowerbinding affinity with one or more flavivirus group-reactive mAbs, due tosubstitution of the flavivirus cross-reactive epitope residue(s), butits binding with one or more DEN-2 virus type-specific mAbs is notaffected.

Specific, non-limiting examples of an isolated polypeptide comprising atleast one flavivirus cross-reactive epitope residue with reduced orablated cross-reactivity include, the amino acid sequences shown in SEQID NO: 16 (G₁₀₄H), SEQ ID NO: 18 (G₁₀₆Q), SEQ ID NO: 20 (L₁₀₇K), SEQ IDNO: 22 (E₁₂₆A), SEQ ID NO: 24 (T₂₂₆N), SEQ ID NO: 26 (W₂₃₁F), SEQ ID NO:28 (W₂₃₁L), SEQ ID NO: 30 (E₁₂₆A/T₂₂₆N), SEQ ID NO: 83, and SEQ ID NO:87.

Manipulation of the nucleotide sequence of a flavivirus cross-reactiveepitope using standard procedures, including for instance site-directedmutagenesis or PCR and M13 primer mutagenesis, can be used to producevariants with reduced or ablated cross-reactivity. Details of thesetechniques are provided in Sambrook et al. (ed.), Molecular Cloning: ALaboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989. The simplest modificationsinvolve the substitution of one or more amino acids for amino acidshaving similar physiochemical and/or structural properties. Theseso-called conservative substitutions are likely to have minimal impacton the activity and/or structure of the resultant protein. Examples ofconservative substitutions are shown below.

Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

Conservative substitutions generally maintain (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Substitutions that should reduce or ablate antibody recognition withoutaltering E-glycoprotein structural conformation, disrupting dimerinteractions, or impairing particle formation, maturation, or secretioninclude: Gly to His, Gly to Gln, Leu to Lys, Glu to Ala, Thr to Asn, Trpto Phe, and Trp to Leu.

The substitutions which in general are expected to produce the greatestchanges in protein properties will be non-conservative, for instancechanges in which (a) a hydrophilic residue, for example, seryl orthreonyl, is substituted for (or by) a hydrophobic residue, for example,leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, for example, lysyl, arginyl, orhistadyl, is substituted for (or by) an electronegative residue, forexample, glutamyl or aspartyl; or (d) a residue having a bulky sidechain, for example, phenylalanine, is substituted for (or by) one nothaving a side chain, for example, glycine.

The disclosure also provides isolated nucleic acids that encode thedescribed polypeptides. Nucleic acids of the invention thus includenucleic acids that encode: 1) polypeptides comprising at least oneflavivirus cross-reactive epitope with reduced or ablatedcross-reactivity; and 2) polypeptides that that are at least 95%identical to the polypeptides comprising at least one flaviviruscross-reactive epitope with reduced or ablated cross-reactivity.

Recombinant nucleic acids may, for instance, contain all or part of adisclosed nucleic acid operably linked to a regulatory sequence orelement, such as a promoter, for instance, as part of a clone designedto express a protein. Cloning and expression systems are commerciallyavailable for such purposes and are well known in the art. Thedisclosure also provides cells or organisms transformed with recombinantnucleic acid constructs that encode all or part of the describedpolypeptides. Also disclosed are virus-like particles (VLPs) thatinclude one or more of the described flavivirus E-glycoproteinpolypeptides.

VI. Specific Binding Agents

This disclosure provides specific binding agents that bind topolypeptides disclosed herein, e.g., flavivirus E-glycoproteinpolypeptides with reduced or ablated cross-reactivity. The binding agentmay be useful for identifying flavivirus cross-reactive epitopes, andfor detecting and purifying polypeptides comprising flaviviruscross-reactive epitopes. Examples of the binding agents are a polyclonalor monoclonal antibody, and fragments thereof, that bind to polypeptidesdisclosed herein. A specific, non-limiting example of a polyclonalantibody is polyclonal anti-DEN-2 MHIAF. Specific, non-limiting examplesof mAbs include 4G2, 6B6C-1, 1B7-5, 10A1D-2, 1A5D-1, and 1B4C-2.

Monoclonal or polyclonal antibodies can be raised to recognize thepolypeptides described herein, or variants thereof. Optimally,antibodies raised against these polypeptides will specifically detectthe polypeptide with which the antibodies are generated. That is,antibodies raised against the polypeptide will recognize and bind thepolypeptide, and will not substantially recognize or bind to otherpolypeptides or antigens. The determination that an antibodyspecifically binds to a target polypeptide is made by any one of anumber of standard immunoassay methods; for instance, the Westernblotting technique (Sambrook et al. (ed.), Molecular Cloning: ALaboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989), Ag-ELISA and IFA.

Substantially pure flavivirus recombinant polypeptide antigens suitablefor use as immunogens can be isolated from the transformed cellsdescribed herein, using methods well known in the art. Monoclonal orpolyclonal antibodies to the antigens can then be prepared.

Monoclonal antibodies to the polypeptides can be prepared from murinehybridomas according to the classic method of Kohler & Milstein (Nature256:495-97, 1975), or a derivative method thereof. Briefly, a mouse isrepetitively inoculated with a few micrograms of the selected proteinimmunogen (for example, a polypeptide comprising at least one flaviviruscross-reactive epitope with reduced or ablated cross-reactivity, aportion of a polypeptide comprising at least one flaviviruscross-reactive epitope with reduced or ablated cross-reactivity, or asynthetic peptide comprising at least one flavivirus cross-reactiveepitope with reduced or ablated cross-reactivity) over a period of a fewweeks. The mouse is then sacrificed, and the antibody-producing cells ofthe spleen isolated. The spleen cells are fused by means of polyethyleneglycol with mouse myeloma cells, and the excess unfused cells destroyedby growth of the system on selective media comprising aminopterin (HATmedia). The successfully fused cells are diluted and aliquots of thedilution placed in wells of a microtiter plate where growth of theculture is continued. Antibody-producing clones are identified bydetection of antibody in the supernatant fluid of the wells byimmunoassay procedures, such as ELISA, as originally described byEngvall (Meth. Enzymol., 70:419-39, 1980), or a derivative methodthereof. Selected positive clones can be expanded and their monoclonalantibody product harvested for use. Detailed procedures for monoclonalantibody production are described in Harlow and Lane, Using Antibodies:A Laboratory Manual, CSHL, New York, 1999.

Polyclonal antiserum containing antibodies can be prepared by immunizingsuitable animals with a polypeptide comprising at least one flaviviruscross-reactive epitope with reduced or ablated cross-reactivity, aportion of a polypeptide comprising at least one flaviviruscross-reactive epitope with reduced or ablated cross-reactivity, or asynthetic peptide comprising at least one flavivirus cross-reactiveepitope with reduced or ablated cross-reactivity, which can beunmodified or modified, to enhance immunogenicity.

Effective antibody production (whether monoclonal or polyclonal) isaffected by many factors related both to the antigen and the hostspecies. For example, small molecules tend to be less immunogenic thanothers and may require the use of carriers and adjuvant. Also, hostanimals vary in response to site of inoculations and dose, with eitherinadequate or excessive doses of antigen resulting in low titerantisera. Small doses (ng level) of antigen administered at multipleintradermal sites appear to be most reliable. An effective immunizationprotocol for rabbits can be found in Vaitukaitis et al. (J. Clin.Endocrinol. Metab., 33:988-91, 1971).

Booster injections can be given at regular intervals, and antiserumharvested when the antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begins to fall. See, forexample, Ouchterlony et al., Handbook of Experimental Immunology, Wier,D. (ed.), Chapter 19, Blackwell, 1973. A plateau concentration ofantibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12μM). Affinity of the antisera for the antigen is determined by preparingcompetitive binding curves, as described, for example, by Fisher (Manualof Clinical Immunology, Ch. 42, 1980).

Antibody fragments may be used in place of whole antibodies and may bereadily expressed in prokaryotic host cells. Methods of making and usingimmunologically effective portions of monoclonal antibodies, alsoreferred to as “antibody fragments,” are well known and include thosedescribed in Better & Horowitz, Methods Enzymol. 178:476-96, 1989;Glockshuber et al., Biochemistry 29:1362-67, 1990; and U.S. Pat. No.5,648,237 (Expression of Functional Antibody Fragments); U.S. Pat. No.4,946,778 (Single Polypeptide Chain Binding Molecules); and U.S. Pat.No. 5,455,030 (Immunotherapy Using Single Chain Polypeptide BindingMolecules), and references cited therein. Conditions whereby apolypeptide/binding agent complex can form, as well as assays for thedetection of the formation of a polypeptide/binding agent complex andquantitation of binding affinities of the binding agent and polypeptide,are standard in the art. Such assays can include, but are not limitedto, Western blotting, immunoprecipitation, immunofluorescence,immunocytochemistry, immunohistochemistry, fluorescence activated cellsorting (FACS), fluorescence in situ hybridization (FISH),immunomagnetic assays, ELISA, ELISPOT (Coligan et al., Current Protocolsin Immunology, Wiley, NY, 1995), agglutination assays, flocculationassays, cell panning, etc., as are well known to one of skill in theart.

Binding agents of this disclosure can be bound to a substrate (forexample, beads, tubes, slides, plates, nitrocellulose sheets, etc.) orconjugated with a detectable moiety, or both bound and conjugated. Thedetectable moieties contemplated for the present disclosure can include,but are not limited to, an immunofluorescent moiety (for example,fluorescein, rhodamine), a radioactive moiety (for example, ³²P, ¹²⁵I,³⁵S), an enzyme moiety (for example, horseradish peroxidase, alkalinephosphatase), a colloidal gold moiety, and a biotin moiety. Suchconjugation techniques are standard in the art (for example, see Harlowand Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999;Yang et al., Nature, 382:319-24, 1996).

VII. Detection of Flavivirus Antibodies

The present disclosure further provides a method of detecting aflavivirus-reactive antibody in a sample, comprising contacting thesample with a polypeptide or peptide of this disclosure under conditionwhereby an antibody/polypeptide complex can form; and detectingformation of the complex, thereby detecting flavivirus antibody in asample.

The method of detecting flavivirus-reactive antibody in a sample can beperformed, for example, by contacting a fluid or tissue sample from asubject with a polypeptide of this disclosure and detecting the bindingof the polypeptide to the antibody. A fluid sample of this method cancomprise any biological fluid which could contain the antibody, such ascerebrospinal fluid, blood, bile plasma, serum, saliva and urine. Otherpossible examples of body fluids include sputum, mucus and the like.

Enzyme immunoassays such as IFA, ELISA and immunoblotting can be readilyadapted to accomplish the detection of flavivirus antibodies accordingto the methods of this disclosure. An ELISA method effective for thedetection of the antibodies can, for example, be as follows: 1) bind thepolypeptide to a substrate; 2) contact the bound polypeptide with afluid or tissue sample containing the antibody; 3) contact the abovewith a secondary antibody bound to a detectable moiety which is reactivewith the bound antibody (for example, horseradish peroxidase enzyme oralkaline phosphatase enzyme); 4) contact the above with the substratefor the enzyme; 5) contact the above with a color reagent; and 6)observe/measure color change or development.

Another immunologic technique that can be useful in the detection offlavivirus antibodies uses mAbs for detection of antibodies specificallyreactive with flavivirus polypeptides in a competitive inhibition assay.Briefly, a sample is contacted with a polypeptide of this inventionwhich is bound to a substrate (for example, a 96-well plate). Excesssample is thoroughly washed away. A labeled (for example, enzyme-linked,fluorescent, radioactive, etc.) mAb is then contacted with anypreviously formed polypeptide-antibody complexes and the amount of mAbbinding is measured. The amount of inhibition of mAb binding is measuredrelative to a control (no antibody), allowing for detection andmeasurement of antibody in the sample. The degree of mAb bindinginhibition can be a very specific assay for detecting a particularflavivirus variety or strain, when based on mAb binding specificity fora particular variety or strain of flavivirus. mAbs can also be used fordirect detection of flavivirus in cells by, for example, IFA accordingto standard methods.

As a further example, a micro-agglutination test can be used to detectthe presence of flavivirus antibodies in a sample. Briefly, latex beads,red blood cells or other agglutinable particles are coated with apolypeptide of this disclosure and mixed with a sample, such thatantibodies in the sample that are specifically reactive with the antigencrosslink with the antigen, causing agglutination. The agglutinatedpolypeptide-antibody complexes form a precipitate, visible with thenaked eye or measurable by spectrophotometer.

In yet another example, a microsphere-based immunoassay can be used todetect the presence of flavivirus antibodies in a sample. Briefly,microsphere beads are coated with a polypeptide of this disclosure andmixed with a sample, such that antibodies in the sample that arespecifically reactive with the antigen bind the antigen. The bead-boundpolypeptide-antibody complexes are allowed to react with fluorescent-dyelabeled anti-species antibody (such as FITC-labeled goat anti-humanIgM), and are measured using a microsphere reader (such as a Luminexinstrument).

The present disclosure further provides a method of diagnosing aflavivirus infection in a subject, comprising contacting a sample fromthe subject with the polypeptide of this disclosure under conditionswhereby an antibody/polypeptide complex can form; and detectingantibody/polypeptide complex formation, thereby diagnosing a flavivirusinfection in a subject.

In examples of the diagnostic methods, the polypeptide of thisdisclosure can be bound to a substrate and contacted with a fluid samplesuch as blood, serum, urine or saliva. This sample can be taken directlyfrom the patient or in a partially purified form. In this manner,antibodies specific for the polypeptide (the primary antibody) willspecifically react with the bound polypeptide. Thereafter, a secondaryantibody bound to, or labeled with, a detectable moiety can be added toenhance the detection of the primary antibody. Generally, the secondaryantibody will be selected for its ability to react with multiple siteson the primary antibody. Thus, for example, several molecules of thesecondary antibodies can react with each primary antibody, making theprimary antibody more detectable.

The detectable moiety allows for visual detection of a precipitate or acolor change, visual detection by microscopy, or automated detection byspectrometry, radiometric measurement or the like. Examples ofdetectable moieties include fluorescein, rhodamine, Cy5, and Cy3 (forfluorescence microscopy and/or the microsphere-based immunoassay),horseradish peroxidase (for either light or electron microscopy andbiochemical detection), biotin-streptavidin (for light or electronmicroscopy) and alkaline phosphatase (for biochemical detection by colorchange).

VIII. Pharmaceutical and Immune Stimulatory Compositions and UsesThereof

Pharmaceutical compositions including flavivirus nucleic acid sequencesor flavivirus polypeptides comprising at least one flaviviruscross-reactive epitope with reduced or ablated cross-reactivity are alsoencompassed by the present disclosure. These pharmaceutical compositionsinclude a therapeutically effective amount of one or more activecompounds, such as flavivirus polypeptides comprising at least oneflavivirus cross-reactive epitope with reduced or ablatedcross-reactivity, or one or more nucleic acid molecules encoding thesepolypeptides, in conjunction with a pharmaceutically acceptable carrier.It is contemplated that in certain embodiments, flavivirus nucleic acidsequences or flavivirus polypeptides comprising multiple flaviviruscross-reactive epitopes with reduced or ablated cross-reactivity will beuseful in preparing the pharmaceutical compositions of the disclosure.

Disclosed herein are substances suitable for use as immune stimulatorycompositions for the inhibition or treatment of a flavivirus infection,for example, a dengue virus infection. In one embodiment, an immunestimulatory composition contains a flavivirus polypeptide including atleast one flavivirus cross-reactive epitope with reduced or ablatedcross-reactivity. In a further embodiment, the immune stimulatorycomposition contains a nucleic acid vector that includes flavivirusnucleic acid molecules described herein, or that includes a nucleic acidsequence encoding at least one flavivirus cross-reactive epitope withreduced or ablated cross-reactivity. In a specific, non-limitingexample, a nucleic acid sequence encoding at least one flaviviruscross-reactive epitope with reduced or ablated cross-reactivity isexpressed in a transcriptional unit, such as those described inpublished PCT Application Nos. PCT/US99/12298 and PCT/US02/10764 (bothof which are incorporated herein in their entirety).

The provided immune stimulatory flavivirus polypeptides, constructs orvectors encoding such polypeptides, are combined with a pharmaceuticallyacceptable carrier or vehicle for administration as an immunestimulatory composition to human or animal subjects. In a particularembodiment, the immune stimulatory composition administered to a subjectdirects the synthesis of a mutant flavivirus E-glycoprotein as describedherein, and a cell within the body of the subject, after incorporatingthe nucleic acid within it, secretes VLPs comprising the mutantE-glycoprotein with reduced or ablated cross-reactivity. It is believedthat such VLPs then serve as an in vivo immune stimulatory composition,stimulating the immune system of the subject to generate protectiveimmunological responses. In some embodiments, more than one immunestimulatory flavivirus polypeptide, construct or vector may be combinedto form a single preparation.

The immunogenic formulations may be conveniently presented in unitdosage form and prepared using conventional pharmaceutical techniques.Such techniques include the step of bringing into association the activeingredient and the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredient with liquid carriers.Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition of asterile liquid carrier, for example, water for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets commonly used by oneof ordinary skill in the art.

In certain embodiments, unit dosage formulations are those containing adose or unit, or an appropriate fraction thereof, of the administeredingredient. It should be understood that in addition to the ingredientsparticularly mentioned above, formulations encompassed herein mayinclude other agents commonly used by one of ordinary skill in the art.

The compositions provided herein, including those for use as immunestimulatory compositions, may be administered through different routes,such as oral, including buccal and sublingual, rectal, parenteral,aerosol, nasal, intramuscular, subcutaneous, intradermal, and topical.They may be administered in different forms, including but not limitedto solutions, emulsions and suspensions, microspheres, particles,microparticles, nanoparticles, and liposomes.

The volume of administration will vary depending on the route ofadministration. By way of example, intramuscular injections may rangefrom about 0.1 ml to about 1.0 ml. Those of ordinary skill in the artwill know appropriate volumes for different routes of administration.

A relatively recent development in the field of immune stimulatorycompounds (for example, vaccines) is the direct injection of nucleicacid molecules encoding peptide antigens (broadly described in Janeway &Travers, Immunobiology: The Immune System In Health and Disease, page13.25, Garland Publishing, Inc., New York, 1997; and McDonnell & Askari,N. Engl. J. Med. 334:42-45, 1996). Vectors that include nucleic acidmolecules described herein, or that include a nucleic acid sequenceencoding a flavivirus polypeptide comprising at least one flaviviruscross-reactive epitope with reduced or ablated cross-reactivity may beutilized in such DNA vaccination methods.

Thus, the term “immune stimulatory composition” as used herein alsoincludes nucleic acid vaccines in which a nucleic acid molecule encodinga flavivirus polypeptide comprising at least one flaviviruscross-reactive epitope with reduced or ablated cross-reactivity isadministered to a subject in a pharmaceutical composition. For geneticimmunization, suitable delivery methods known to those skilled in theart include direct injection of plasmid DNA into muscles (Wolff et al.,Hum. Mol. Genet. 1:363, 1992), delivery of DNA complexed with specificprotein carriers (Wu et al., J. Biol. Chem. 264:16985, 1989),co-precipitation of DNA with calcium phosphate (Benvenisty and Reshef,Proc. Natl. Acad. Sci. 83:9551, 1986), encapsulation of DNA in liposomes(Kaneda et al., Science 243:375, 1989), particle bombardment (Tang etal., Nature 356:152, 1992; Eisenbraun et al., DNA Cell Biol. 12:791,1993), and in vivo infection using cloned retroviral vectors (Seeger etal., Proc. Natl. Acad. Sci. 81:5849, 1984). Similarly, nucleic acidvaccine preparations can be administered via viral carrier.

The amount of immunostimulatory compound in each dose of an immunestimulatory composition is selected as an amount that induces animmunostimulatory or immunoprotective response without significant,adverse side effects. Such amount will vary depending upon whichspecific immunogen is employed and how it is presented. Initialinjections may range from about 1 μg to about 1 mg, with someembodiments having a range of about 10 μg to about 800 μg, and stillother embodiments a range of from about 25 μg to about 500 μg. Followingan initial administration of the immune stimulatory composition,subjects may receive one or several booster administrations, adequatelyspaced. Booster administrations may range from about 1 μg to about 1 mg,with other embodiments having a range of about 10 μg to about 750 μg,and still others a range of about 50 μg to about 500 μg. Periodicboosters at intervals of 1-5 years, for instance three years, may bedesirable to maintain the desired levels of protective immunity.

It is also contemplated that the provided immunostimulatory moleculesand compositions can be administered to a subject indirectly, by firststimulating a cell in vitro, which stimulated cell is thereafteradministered to the subject to elicit an immune response. Additionally,the pharmaceutical or immune stimulatory compositions or methods oftreatment may be administered in combination with other therapeutictreatments.

IX. Kits

Also provided herein are kits useful in the detection and/or diagnosisof flaviviruses. An example of an assay kit provided herein is arecombinant flavivirus polypeptide (or fragment thereof) as an antigenand an enzyme-conjugated anti-human antibody as a second antibody.Examples of such kits also can include one or more enzymatic substrates.Such kits can be used to test if a sample from a subject containsantibodies against a flavivirus-specific protein. In such a kit, anappropriate amount of a flavivirus polypeptide (or fragment thereof) isprovided in one or more containers, or held on a substrate. A flaviviruspolypeptide can be provided in an aqueous solution or as a freeze-driedor lyophilized powder, for instance. The container(s) in which theflavivirus polypeptide(s) are supplied can be any conventional containerthat is capable of holding the supplied form, for instance, microfugetubes, ampoules, or bottles.

The amount of each polypeptide supplied in the kit can be anyappropriate amount, and can depend on the market to which the product isdirected. For instance, if the kit is adapted for research or clinicaluse, the amount of each polypeptide provided would likely be an amountsufficient for several assays. General guidelines for determiningappropriate amounts can be found, for example, in Ausubel et al. (eds.),Short Protocols in Molecular Biology, John Wiley and Sons, New York,N.Y., 1999 and Harlow and Lane, Using Antibodies: A Laboratory Manual,CSHL, New York, 1999.

The subject matter of the present disclosure is further illustrated bythe following non-limiting Examples.

EXAMPLE 1 Identification of DII Cross-Reactive Epitope Residues

This example demonstrates the identification of flaviviruscross-reactive epitopes using a structure-based rational mutagenesismethod.

Cell Culture, Virus Strain and Recombinant Plasmid

COS-1 cells (ATCC CRL 1650; Manassas, Va.) were grown at 37° C. with 5%CO₂ on Dulbecco's modified Eagle's minimal essential medium (DMEM,GIBCO, Grand Island, N.Y.) supplemented with 10% heat-inactivated fetalbovine serum (FBS, Hyclone Laboratories, Inc., Logan, Utah), 110 mg/lsodium pyruvate, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 20ml/17.5% NaHCO₃, 100 U/ml penicillin, and 100 μg/ml streptomycin. CHOcells (ATCC CCL 61; Manassas, Va.) were grown under the same conditionsas COS-1 cells with DMEM/F12 nutrient mixture (GIBCO, Grand Island,N.Y.).

Flavivirus plasmids capable of expressing extracellular VLPs composed ofprM/M and E-glycoproteins for JE, WN, SLE, and the four DEN virusserotypes have been constructed (Chang et al., J. Virol. 74:4244-52,2000; Chang et al., Virology 306:170-80, 2003; Davis et al., J. Virol.75:4040-47, 2001). These VLPs, produced by recombinantplasmid-transformed eukaryotic cells, contain the flavivirus prM/M andE-glycoproteins in their native viral conformations, and althoughnon-infectious, they maintain many of the same properties as maturevirus particles including, hemagglutination activity, membrane fusion,and the induction of protective immune responses in animals (Chang etal., J. Virol. 74:4244-52, 2000; Chang et al., Virology 306:170-80,2003; Davis et al., J. Virol. 75:4040-47, 2001; Hunt, et al., J. Virol.Methods 97:133-49, 2001).

The recombinant expression plasmid pCB8D2-2J-2-9-1 (the DEN-2 prM/Eexpression plasmid, Chang et al, Virology 306:170-80, 2003) was used asthe template DNA for both site-directed mutagenesis and for transientexpression of DEN-2 recombinant antigen (see below). This plasmidincludes the human cytomegalovirus early gene promoter, Kozak sequence,JE virus signal sequence, DEN-2 virus prM/M gene, DEN-2 virus chimeric Egene (with amino-terminal 80% from DEN-2 virus and carboxy-terminal 20%from JE virus), and bovine growth hormone poly(A) signal. Thereplacement of the terminal 20% of DEN-2 virus E gene sequences with JEvirus E gene sequences dramatically increases the secretion ofextracellular VLPs into the culture medium without altering the nativeDEN-2 virus E-glycoprotein conformation (Chang et al., Virology306:170-80, 2003).

Procedural Algorithm

To localize the epitopes responsible for inducing flaviviruscross-reactive antibodies, the following procedural algorithm wasdeveloped: Strictly-conserved flavivirus residues were initiallyidentified. These residues were mapped onto the 2.0 Å resolutionE-glycoprotein structure for TBE virus (Rey et al., Nature 375:291-98,1995) and onto a computer predicted homology model structure for theDEN-2 virus E-glycoprotein using the Swiss-Pdb Viewer 3.7 structureanalysis software (Guex et al., Electrophoresis 18:2714-23, 1997;available on the ExPASy Molecular Biology Server). A brief review ofhigh resolution structures for antigen-antibody complexes revealed that10-20 residues typically are involved in making direct contacts betweenthe antigen epitope and antibody paratope. These contacts result in20-30 residues that are “buried” by the typical antibody footprint,measuring approximately 20×30 Å. On average however, only 25% of theburied side chains, or 4-6 residues, account for most of the mAb bindingenergy (Arevalo et al., Nature 356:859-63, 1993; Bhat et al., PNAS91:1089-93, 1994; Davies & Cohen, PNAS 93:7-12, 1996; Faebler et al., J.Mol. Biol. 313:83-97, 2001; Fleury et al., Nature St. Biol. 6:530-34,1999; Li et al., Biochemistry 39:6296-6309, 2000; Lo et al., J. Mol.Biol. 285:2177-98, 1999; and Mylvaganam et al., J. Mol. Biol.281:301-22, 1998).

The following criteria were developed to select probable flavivirusgroup cross-reactive epitope residues: 1) an amino acid located in DII(for example, amino acids 52-135 and 195-285 in the TBE virusE-glycoprotein (Rey et al., Nature 375:291-98, 1995); 52-132 and 193-280in the DEN-2 virus E-glycoprotein (Modis et al., PNAS 100:6986-91,2003)) and conserved among more than one flavivirus; 2) amino acids onthe outer or lateral surface of the E-glycoprotein dimer; 3) amino acidswith at least 35% surface accessibility potential; 4) side chainprojections accessible to antibody paratopes; and 5) residues with hightemperature (β-) factors should be favored, as these residues tend to beflexible and are able to conform to the antibody paratope, increasingthe antibody-antigen affinity.

Using this structure-based design approach, candidate flaviviruscross-reactive epitope residues were narrowed down from a total of 53conserved amino acids in DII (38 invariant and 15 almost completelyconserved), to less than ten probable DII cross-reactive epitoperesidues. Amino acid substitutions at these probable cross-reactiveepitope residues were computer modeled, selecting substitutions thatshould reduce or ablate antibody recognition without alteringE-glycoprotein structural conformation, disrupting dimer interactions,or impairing particle formation, maturation, or secretion. For thisreason, cysteine residues otherwise satisfying the cross-reactiveepitope criteria were not considered for mutagenesis because of theirinvolvement in disulphide bridging necessary for proper E-glycoproteinstructure and function (Modis et al., PNAS 100:6986-91, 2003; Rey etal., Nature 375:291-98, 1995).

Stability calculations were performed for all possible amino acidsubstitutions of candidate residues using the FOLD-X server (Guerois etal., J. Mol. Biol. 320:369-87, 2002; available on the internet) and theTBE virus E-glycoprotein pdb file coordinates (Rey et al., Nature375:291-98, 1995). Amino acid substitutions modeled in the TBE virusE-glycoprotein with free energies of folding equal to or less than thatof the non-mutated wild-type E-glycoprotein were re-examined with theSwiss-PdbViewer software to identify those substitutions that minimizedlocal structural disturbances while maintaining structurally relevantbiochemical interactions such as hydrogen bonding and/or chargeinteractions with neighboring amino acids. Because the outer surface ofmature flavivirus particles are covered in a dense network of E andprM/M proteins, any conformational changes in the E-glycoprotein arelikely to induce concerted reorganization across the surface of thevirion (Kuhn et al., Cell 108:717-25, 2002; Modis et al., PNAS100:6986-91, 2003). A comparison of the a priori stability calculationsbased on the TBE virus E-glycoprotein structure with a posterioristability calculations from the DEN-2 virus atomic structure are shownin Table 2.

Site-Directed Mutagenesis

Site-specific mutations were introduced into the DEN-2 virus E geneusing the Stratagene Quick Change® multi site-directed mutagenesis kit(Stratagene, La Jolla, Calif.) and pCB8D2-2J-2-9-1 as DNA templatefollowing the manufacturer's recommended protocols. The sequences of themutagenic primers used for all constructs are listed in Table 1. Four orfive colonies from each mutagenic PCR transformation were selected andgrown in 5 ml LB broth cultures, mini-prepped and sequenced. Structuralgene regions and regulatory elements of all purified plasmids weresequenced entirely upon identification of the correct mutation.Automated DNA sequencing was performed using a Beckman Coulter CEQ™ 8000genetic analysis system (Beckman Coulter, Fullerton, Calif.) andanalyzed using Beckman Coulter CEQ™ 8000 (Beckman Coulter, Fullerton,Calif.) and Lasergene® software (DNASTAR, Madison, Wis.).

Transient Expression of DEN-2 Virus Recombinant Antigens in COS-1 or CHOCells

COS-1 and CHO cells were electroporated with pCB8D2-2J-2-9-1 using theprotocol described by Chang et al. (J. Virol. 74:4244-52, 2000).Electroporated cells were recovered in 50 ml DMEM, seeded into 150 cm²culture flasks for VLP expression and into 96 well tissue culture plates(Costar® #3603; Corning, Inc., Corning, N.Y.) for IFA, and incubated at37° C. with 5% CO₂. Six to eight hours following electroporation, thegrowth medium in the 150 cm² culture flasks was replaced with DMEMcontaining 2% FBS. Cells in 96 well plates for IFA were fixed 14-18hours post electroporation. Tissue-culture medium and cells wereharvested 48 and 96 hours post electroporation for antigencharacterization.

Characterization of Mutant pCB8D2-J2-2-9-1 Infected Cells and SecretedAntigen

Fourteen to eighteen hours following electroporation, 96 well tissueculture plates containing cells transformed with the mutatedpCB8D2-2J-2-9-1 clones were washed twice with phosphate buffered saline(PBS), fixed with 3:1 acetone:PBS for 10 minutes and air dried.E-glycoprotein-specific InAbs specific for each of the threeE-glycoprotein domains were used to determine affinity reductions in DIIcross-reactive epitopes by indirect IFA as described by Chang et al. (J.Virol. 74:4244-52, 2000).

Tissue culture medium was harvested 48 hours and 96 hours followingelectroporation. Cell debris was removed from tissue culture media bycentrifugation for 30 minutes at 10,000 rpm. Ag-ELISA was used to detectsecreted antigen from the mutagenized pCB8D2-2J-2-9-1 transformed COS-1cells. Secreted antigen was captured with polyclonal rabbit anti-DEN-2sera (Roehrig et al., Virology 246:317-28, 1998) at a 1:10,000 dilution.Murine hyper-immune ascetic fluid (MHIAF) specific for DEN-2 virus wasused at a 1:3000 dilution to detect captured antigen, and this MHIAF wasdetected using horseradish peroxidase conjugated goat anti-mouse HIAF ata 1:5000 dilution. Secreted antigen from tissue culture medium wasconcentrated by centrifugation overnight at 19,000 rpm, and resuspendedin TNE buffer (50 mM Tris, 100 mM NaCl, 10 mM EDTA, pH 7.5) to1/200^(th) the original volume. Concentrated antigen was analyzed with apanel of anti-DEN-2 mAbs in Ag-ELISA to determine mAb end pointreactivities of the mutated antigens following the protocol of Roehriget al. (Virology 246:317-28, 1998).

Affinity Reductions in DII Cross-Reactive Epitopes

Three anti-DEN-2 mAbs, 4G2, 6B6C-1 and 1B7-5, were used to determineaffinity reductions in DII cross-reactive epitopes. These three mAbsshare several characteristics: they recognize surface accessibleepitopes in DII, they are flavivirus group- or subgroup-reactive, theyare reduction-denaturation sensitive, they block virus-mediatedcell-membrane fusion, they neutralize virus infectivity, and trypticfragment mapping indicates that the binding domains of these three mAbsare formed by two discontinuous DEN-2 virus E-glycoprotein peptidefragments, aa1-120 and 158-400 (Aaskov et al., Arch Virol. 105:209-21,1989; Henchal et al., Am. J. Trop. Med. Hyg. 34:162-69, 1985; Megret etal., Virology 187:480-91, 1992; Roehrig et al., Virology 246:317-28,1998). Prospective cross-reactive epitope residues were assessed bylooking for decreases in the reactivity of these three DII flaviviruscross-reactive mAbs for the mutant plasmid transfected cells by IFA, andmutant VLPs in Ag-ELISA. Proper E-glycoprotein folding and structuralconformation was assessed with a panel of E-glycoprotein DEN viruscomplex-, subcomplex-, and type-specific mAbs.

Four potential flavivirus cross-reactive epitope residues were initiallyfocused on. Single amino acid substitutions were introduced into theDEN-2 prM/E expression plasmid at the following positions (of SEQ ID NO:14): Gly₁₀₆ to Glu (G₁₀₆Q), Trp₂₃₁ to Phe (W₂₃₁F), His₂₄₄ to Arg(H₂₄₄R), and Lys₂₄₇ to Arg (K₂₄₇R) (Table 1). Substitutions at Gly₁₀₆and Trp₂₃₁ strongly interfered with the binding of flaviviruscross-reactive mAbs (Table 3). However, substitutions at His₂₄₄ andLys₂₄₇ did not have a measurable effect on the binding of the crossreactive mAbs or of any other mAbs from the panel.

Gly₁₀₆ is located within the fusion peptide at the very tip of DII inthe E-glycoprotein monomer (Allison et al., J. Virol. 73:5605-12, 1999;FIGS. 1 and 2). As with the other fusion peptide residues, Gly₁₀₆ isstrongly conserved across the flaviviruses, the one exception beingModoc virus with alanine at this position (Table 4). Gly₁₀₆ is locatedat the distal end of each E-monomer along the upper and outer-lateralsurface of the dimer. This residue has moderately high surfaceaccessibility, and its relatively high temperature (P—) factor suggestsits potential flexibility. The substitution of a large, bulky, polarglutamine for the glycine at this position was modeled. The glutaminesubstitution fit well into the surrounding region, did not appear todisrupt the local hydrogen bonding network, and produced acceptablestability calculations using the TBE virus E-glycoprotein structurecoordinates (Table 2).

Trp₂₃₁ is located in a long intervening loop sequence between DIIβ-strands h and i (Modis et al., PNAS 100:6986-91, 2003; FIG. 1). Trp₂₃₁lays in a trough on the upper and outer surface of DII (FIG. 2). It isstructurally close to the glycan on Asn₆₇, and lies laterally exteriorto the disulfide bridge between Cys₆₀ and Cys₁₂₁. The large hydrophobicside chain lays parallel to the dimer surface within this trough. Thisresidue is only moderately surface accessible yet its high temperature(β-) factor and the lack of hydrogen bonding from surrounding residuesto the side chain suggest its potential flexibility. Although all of thesubstitutions that were modeled at Trp₂₃₁ were predicted to inducesubstantially high energetic costs from the stability analyses, thephenylalanine substitution was the least costly substitution at thisposition (Table 2). The phenylalanine fit well into the surroundingmolecular region with limited disruption of the local hydrogen bondingnetwork.

Binding of the G₁₀₆Q mutant to either of the two flavivirusgroup-reactive mAbs, 4G2 and 6B6C-1, was not detected (Table 3). DEN-2type-specific mAbs 1A5D-1 and 1B4C-2 (DII and DI, respectively)exhibited reduced affinities for G₁₀₆Q transfected cells and forsecreted VLPs. Dengue complex-specific mAb 10A/D-2 also exhibitedmoderately reduced reactivity for the G₁₀₆Q VLP antigen (Table 3).However, the reactivity of the G₁₀₆Q mutant was unchanged from thereactivity of the wild-type pCB8D2-2J-2-9-1 antigen for polyclonalanti-DEN-2 MHIAF, as well as for the remaining subcomplex- andtype-specific mAbs: 9A4D-1 (DI), 4E5 (DII), and 3H5, 9A3D-8, 10A4D-2,9D12, and 1A1D-2 (DIII) (Table 3).

The W₂₃₁F substitution also abolished the binding of both flavivirusgroup-reactive mAbs, 4G2 and 6B6C-1, as well as that of flavivirussubgroup-reactive mAb 1B7-5 (Table 3). This substitution additionallyinterfered with the binding of type-specific DI mAb 1B4C-2, but thebinding of the remaining subcomplex- and type-specific DI, DII and DIIImAbs and a polyclonal DEN-2 MHIAF were unchanged relative to thenon-mutated wild-type plasmid (Table 3). In three separate experiments,secretion of W₂₃₁F VLP antigen into the tissue culture medium fromtransiently transfected COS-1 cells was not detected. Consequently, theeffects of this substitution could only be analyzed by IFA of plasmidtransfected cells.

The H₂₄₄R and K₂₄₇R substitutions did not have an effect on the bindingof any mAbs in either IFA of transfected cells, or in Ag-ELISA ofsecreted VLP antigen.

EXAMPLE 2 Identification of Additional Cross-Reactive Epitopes ThroughNearest Neighbor Search

This example demonstrates the identification of additionalcross-reactive epitopes using a “nearest neighbor” search.

Following the identification of cross-reactive epitope residues G₁₀₆ andW₂₃₁ (DEN-2 numbering), the E-glycoprotein atomic structure wasreexamined to search for additional flavivirus cross-reactive epitoperesidues. A “nearest neighbor” search was conducted of the surface ofthe E-glycoprotein structure, looking for additional residues locatedwithin 10-15 Å of the identified residue. This distance is within thebinding footprint of a single antibody paratope (Faebler et al., J. Mol.Biol. 313:83-97, 2001). In this second iteration of cross-reactiveepitope residue identification the same five optimality criterion asabove were used, with one change. The criterion of strict conservationacross the flaviviruses was relaxed to now include variable residues. Inthis way, residues either conserved in their physiochemical natureand/or conserved only within a particular flavivirus complex (such asthe four DEN virus serotypes) could be identified.

This nearest neighbor search yielded another seven potentialcross-reactive epitope residues. Amino acid substitutions at thesepositions were modeled into the TBE virus E-glycoprotein structure asdescribed above. Mutagenic PCR primers were then synthesized (Table 1)and used to introduce mutations into the wild-type DEN-2 prM/Eexpression plasmid. Plasmids were transiently transfected into CHOcells, and transfected cells and secreted VLP antigen were analyzed withthe anti-DEN-2 mAb panel (Table 3). The substitutions introduced atthese positions (of SEQ ID NO: 14) were: Lys₆₄ to Asn (K₆₄N), Thr₇₆ toMet (T₇₆M), Gln₇₇ to Arg (Q₇₇R), Gly₁₀₄ to His (G₁₀₄H), Leu₁₀₇ to Lys(L₁₀₇K), Glu₁₂₆ to Ala (E₁₂₆A), and Thr₂₂₆ to Asn (T₂₂₆N) (Table 2). Asingle double mutant combining substitutions at positions 126 and 226(E₁₂₆A/T₂₂₆N) was also examined. Since the initial W₂₃₁F substitutioninterfered with antigen secretion, the effects of an alternativesubstitution at this position, Trp₂₃₁ to Leu (W₂₃₁L), were alsoexamined.

The G₁₀₄H, L₁₀₇K, and W₂₃₁L substitutions had the greatest effect ondecreasing the reactivities of DII cross-reactive mAbs. Gly₁₀₄ islocated on the upper surface of the dimer at the tip of the tight loopstructure which the fusion peptide adopts in the E-glycoprotein dimer(Modis et al., PNAS 100:6986-91, 2003; FIG. 2). The residue hasmoderately high surface accessibility and a relatively high temperature(β-) factor. The replacement of this small aliphatic glycine was modeledwith a large polar histidine at this position. The histidine residuefits well into this pocket and was predicted not to alter thehydrogen-bond network in the region. Moreover, because the tick-borneflaviviruses have a histidine at this position (Table 4) it seemedprobable that this substitution would not disrupt the structure in thislocalized region or elsewhere within DII. In fact, a posterioristability calculations based upon the DEN-2 E atomic structure (Modis etal., PNAS 100:6986-91, 2003) indicate that the G₁₀₄H substitution isenergetically favorable (Table 2).

The G₁₀₄H substitution, like both substitutions examined at Trp₂₃₁,produced a plasmid that was unable to secrete measurable VLP antigeninto the tissue culture medium upon transfection of either COS-1 or CHOcells. Consequently, the effects of G₁₀₄H and W₂₃₁L substitutions wereanalyzed solely by IFA of plasmid transfected cells, as described abovefor W₂₃₁F. The G₁₀₄H substitution ablated the reactivity of all three ofthe flavivirus cross-reactive mAbs, 4G2, 6B6C-1, and 1B7-5. Thetype-specific DII mAb 1A5D-1 also showed strongly reduced reactivity forcells transiently transcribed with this plasmid Table 3). W₂₃₁L showed areduction in mAb reactivities very similar to W₂₃₁F, knocking out anydiscernable recognition of all three cross-reactive mAbs (Table 3). Thereactivity of DI mAb 1B4C-2 was also reduced by this mutation, but therewere no discernable changes in the reactivities of the remainingsubcomplex- and type-specific mAbs or the anti-DEN-2 MHIAF for eitherthe G₁₀₄H or W₂₃₁L plasmid constructs (Table 3).

The L₁₀₇K substituted plasmid exhibited a pattern of reducedreactivities for flavivirus cross-reactive mAbs unlike any of the othersubstitutions. Leu₁₀₇ sits directly below Gly₁₀₆ on the outer lateralsurface of the E-protein dimer. This residue has relatively high surfaceaccessibility and temperature (β-) factor, and its hydrophobicside-chain is directed laterally away from the dimer. This residue isalso strongly conserved across the flaviviruses; the exceptions beingthe tick-borne Powassan virus, JE virus strain SA-14-14-2, and DEN-2virus strain PUO-280 (Table 4). All of these viruses have aphenylalanine instead of a leucine at this position. A large basiclysine was substituted for the leucine at this position. Modeling ofthis L₁₀₇K substitution indicated that it too was unlikely to alter thelocalized hydrogen bonding network. This observation and the lowthermodynamic free energy (ddG) stability calculation (Table 2)suggested that this substitution was unlikely to induce localized ordomain associated conformational changes.

Flavivirus group-reactive mAb 4G2 showed no discernable reactivity forthis construct in either IFA of plasmid transfected cells, or byAg-ELISA of secreted VLP antigen. However, the reactivities of the othertwo cross-reactive mAbs, 6B6C-1 and 1B7-5, were unchanged for thisconstruct relative to the non-mutated wild-type plasmid (Table 3). L₁₀₇Kplasmid-transfected cells and secreted VLP antigen also showedmoderately reduced reactivity for mAbs 1A5D-1, 10A1D-2 and 1B4C-2, whileall other mAbs and the polyclonal MHIAF reactivities were notsignificantly different than they were for the wild-type plasmid (Table3).

Unlike Leu₁₀₇, Glu₁₂₆ appears to be incorporated into epitopesrecognized by flavivirus group-reactive mAb 6B6C-1 and subgroup-reactivemAb 1B7-5, but not in the epitope of flavivirus group-reactive mAb 4G2.Glu₁₂₆ is located 10-12 Å from Trp₂₃₁ in the same trough on the upperand outer surface of DII. The bulky side chain projects from theα-carbon backbone up into this trough producing a moderately highsurface accessibility and a high β-factor (FIG. 2). The replacement ofthis large, negatively charged acidic glutamine was modeled with a smallhydrophobic alanine at this position. This substitution was predicted toinduce a moderately high, but acceptable energetic cost in the freeenergy stability analysis based on the TBE virus E-glycoproteinstructure coordinates (TBE virus equals Lys₁₂₆, Table 2).

The E₁₂₆A substitution reduced the reactivity of flavivirusgroup-reactive mAb 6B6C-1, and moderately reduced the reactivity ofsubgroup-reactive mAb 1B7-5 (Table 3). However, mAb 6B6C-1 exhibitedreduced reactivity only by IFA of mutant plasmid transfected cells, and1B7-5 only showed reactivity reductions for this construct in Ag-ELISA(Table 3). Similarly, type-specific DII mAb 1A5D-1 exhibited moderatelyreduced reactivity by Ag-ELISA, but there was no detectable reduction inits reactivity by IFA (Table 3). The T₂₂₆N substitution did not alterthe reactivity of any of the flavivirus group-reactive mAbs relative tothe non-mutated wild-type plasmid, and the E₁₂₆A/T₂₂₆N double mutantgenerally showed a similar pattern of reduction of mAb reactivity as didE₁₂₆A alone. The two exceptions to this correlation were in thereactivities of mAbs 1B7-5 and 10A1D-2. E₁₂₆A/T₂₂₆N exhibited the samemoderate 87% reduction in Ag-ELISA reactivity for flavivirussubgroup-reactive mAb 1B7-5 as did E₁₂₆A. However, the double mutantalso exhibited a strong 97% reduction for this same mAb by IFA, whichwas not observed for either single mutant (Table 3). DEN viruscomplex-specific mAb 10A1D-2 also exhibited moderate reactivitydecreases by IFA for this double mutant (Table 3).

K₆₄N, T₇₆M, and Q₇₇R were all unchanged in their reactivities for theflavivirus cross-reactive mAbs. The T₇₆M VLP antigen did however showreduced reactivity for DII type-specific mAb 1A5D-1 and for DI mAb1B4C-2 in Ag-ELISA (Table 3).

EXAMPLE 3 Spatial Characterization and Organization of FlavivirusGroup-Reactive Epitope Residues

This example describes the spatial characterization and organization ofexemplary flavivirus cross-reactive epitope residues.

The six residues (G₁₀₄, G₁₀₆, L₁₀₇, E₁₂₆, T₂₂₆, and W₂₃₁) identified asparticipating in the flavivirus cross-reactive epitopes are spatiallyarranged on the DEN-2 virus E-glycoprotein surface in two clusters (FIG.1). The most prominent grouping of these residues is the clustering ofthree residues from the highly conserved fusion peptide region of DII(Allison et al., J. Virol. 75:4268-75, 2001). These residues, Gly₁₀₄,Gly₁₀₆, and Leu₁₀₇, are almost completely conserved across theflaviviruses (Table 4).

The cross-reactive mAbs most strongly affected by substitutions in thisregion were 4G2 and 6B6C-1. These two mAbs are considered to be quitesimilar; both are flavivirus group-reactive and have been grouped intothe A1 epitope of the E-glycoprotein (Gentry et al., Am. J. Trop. Med.Hyg. 31:548-55, 1982; Henchal et al., Am. J. Trop. Med. Hyg. 34:162-69,1985; Mandl et al., J. Virol. 63:564-71, 1989; Roehrig et al., Virology246:317-28, 1998). The data disclosed herein demonstrate that althoughthe epitopes of these two mAbs spatially overlap, they do not containexactly the same residues. Substitutions at G₁₀₄, G₆₀₆, or L₁₀₇ knockout the ability of mAb 4G2 to bind to the E-glycoprotein. However, onlysubstitutions at G₁₀₄ and G₁₀₆ interfere with the binding ability of mAb6B6C-1. L₁₀₇ is therefore not a component of the flavivirusgroup-reactive epitope recognized by mAb 6B6C-1.

The G₁₀₄H substitution dramatically reduced the reactivities of allthree of the flavivirus cross-reactive mAbs for this construct (Table3). Without being bound by theory, it is unlikely that a glycineresidue, with no side chain, would directly participate in the bindingenergetics of an antibody-antigen (Ab-Ag) interaction. However, if aglycine residue is included in the buried surface area of this antibodyepitope, the introduction of a large bulky hydrophobic side chain islikely to disrupt the Ab-Ag shape complementarity and hence increase thedissociation rate-constant (K_(d)) of the Ab-Ag interaction (Li et al.,Nature Struct. Biol. 10:482-88, 2003). G₁₀₄H also reduced therecognition of type-specific DII mAb 1A5D-1 (Table 3). The 1A5D-1epitope is non-neutralizing, reduction sensitive and moderately surfaceaccessible (Roehrig et al., Virology 246:317-28, 1998). All of thefusion peptide substitutions introduced into this region reduced thereactivity of 1A5D-1, consistent with the interpretation that the buriedsurface area footprint of this mAb not only includes DEN-2 virusserotype-specific residues, but also includes these strongly conservedresidues as well. A comparison of the DEN-2 atomic structure withflavivirus E-glycoprotein alignments identifies at least two uniqueDEN-2, DII, surface accessible residues (Glu₇₁ and Asn₈₃), and a thirdresidue variable within DEN-2 but distinct from the other DEN virusserotypes (Thr₈₁). All of these residues are within 10-22 Å of Gly₁₀₄, adistance well within the buried surface area of a typical Ab-Aginterface (Lo et al., J. Mol. Biol. 285:2177-98, 1999). Alternatively,less surface accessible type-specific residues nearby could participatein mAb 1A5D-1 binding since this epitope itself is only moderatelysurface accessible (Roehrig et al., Virology 246:317-28, 1998). Sincethis mAb is DEN-2 virus specific, these type-specific residues would beexpected to provide the majority of the binding energy for 1A5D-1.

The G₁₀₆Q substitution also knocked out the reactivities of both of theflavivirus group-reactive mAbs, 4G2 and 6B6C-1, though it did not alterthe binding of subgroup-reactive mAb 1B7-5 (Table 3, FIG. 2).Type-specific DII mAb 1A5D-1 again lost all measurable reactivity to theG₁₀₆Q construct, as did 1B4C-2. The 1A5D-1 epitope footprint appears toinclude conserved fusion peptide residues in addition to DEN-2serotype-specific residues as discussed herein. The reduced reactivityof DI mAb 1B4C-2 for the G₁₀₆Q construct is difficult to explain.Because of the lack of biological activity of DI, epitope assignments tothis domain can be problematic (Roehrig et al., Virology 246:317-28,1998). Without being bound by theory, the involvement of Gly₁₀₆ as wellas that of Leu₁₀₇ are consistent with the possibility that either theprevious DI assignment is incorrect, or that the 1B4C-2 mAb footprintincludes residues from both DI and DII. However, if 1B4C-2 recognizessuch an inter-domain epitope, this high affinity mAb would be expectedto interfere with the E-glycoprotein dimer to trimer reorganizationassociated with virus-mediated membrane fusion, which it does not.

Leu₁₀₇ is the third residue identified in the fusion peptide region ofDII that is incorporated into flavivirus cross-reactive epitopes. Unlikethe substitutions at E-glycoprotein positions 104 and 106, the L₁₀₇Ksubstitution knocked out the reactivity of flavivirus group-reactive mAb4G2, but it did not alter the reactivity of the other flavivirusgroup-reactive mAb, 6B6C-1 (Table 3, FIG. 2). Beyond this discrepancy,the reactivity patterns of the rest of the mAbs for this construct weresimilar to that observed for the other fusion peptide substitutions.mAbs 1A5D-1, 10A1D-2, and 1B4C-2 all showed little to no reactivity forthe L₁₀₇K construct (Table 3).

Previous studies have examined the effects of mutagenesis in this fusionpeptide region. Pletnev et al. (J. Virol. 67:4956-63, 1993) performedmutagenesis to fusion peptide residues 104 and 107 in a chimericinfectious clone containing the TBE virus structural genes and DEN-4virus non-structural genes. TBE virus has a histidine at position 104 asdo all of the tick-borne flaviviruses. Pletnev et al. constructed theopposite substitution that was constructed herein, H₁₀₄G, replacing thetick-associated histidine with the mosquito-associated glycine, but theywere unable to recover live virus from this construct. They alsoconstructed a double mutant H₁₀₄G/L₁₀₇F from which they were able torecover virus; however, they were unable to detect any effect of thesemutations on mouse neurovirulence. Allison et al. (J. Virol. 75:4268-75,2001) also performed mutagenesis at Leu₁₀₇ examining the role of thisresidue in virus-mediated membrane fusion using TBE virus VLPs. Theyreplaced Leu₁₀₇ with phenylalanine, threonine, or aspartic acid. Theyfound that all of these mutations reduced the rate of fusion. Moreover,consistent with the results presented herein, they found that the L₁₀₇Dsubstitution appeared to completely abolish the binding of their DIIflavivirus group-reactive mAb A1.

The fourth residue identified as having a major effect on the flaviviruscross-reactive mAbs was Trp₂₃₁, an invariant residue across theflaviviruses (Table 4). Both substitutions introduced at Trp₂₃₁dramatically reduced the reactivity of all three of the flaviviruscross-reactive mAbs, 4G2, 6B6C-1, and 1B7-5. This residue isstructurally distant from the fusion peptide region (FIGS. 1 and 2). Itis somewhat surprising that substitutions at this residue affect thebinding of mAbs also shown to recognize the distant fusion peptideresidues. With out being bound by theory, the strict conservation oftryptophan (Table 4) and the predicted high energetic costs ofsubstitutions at this position (Table 2) suggest that this residue couldbe important for proper DI/DII conformational structure and function. Ifthis were the case, the loss of reactivity of mAbs recognizing fusionpeptide residues could occur from the induction of localized structuraldisturbances across DII occurring at a distance from Trp₂₃₁. However,the Trp₂₃₁ substitutions did not significantly affect the binding of anyof the remaining DII mAbs, 4E5, 1A5D-1, and 10A1D-2 (DI or DII); whereasmAb 1A5D-1 reactivity was reduced or ablated by all of the fusionpeptide substitutions. mAb 4E5 does not recognize native virus yet itblocks virus-mediated cell-membrane fusion, presumably by recognizing anepitope that is exposed only during or after low-pH-catalyzedconformational changes (Roehrig et al., Virology 246:317-28, 1998).Without being bound by theory, if substitutions at Trp₂₃, induced domainwide structural alterations, a loss of reactivity of mAb 1A5D-1 (and thepossible exposure of the non-native-accessible mAb 4E5 epitope,resulting in an increase, or at least a change in, the reactivity of mAb4E5 by IFA for these constructs), would be expected. Moreover, thereactivities of polyclonal MHIAF and of all of the DIII mAbs were nodifferent for these constructs than they were for the non-mutatedwild-type plasmid transfected cells (Table 3). DIII however, isreduction-denaturation stable and folds into its native IgC likeconformation even when this domain is expressed alone without theremainder of the E-glycoprotein (Bhardwaj et al., J. Virol. 75:4002-07,2001).

Both W₂₃₁F and W₂₃₁L plasmids, as well as the G₁₀₄H plasmid, failed tosecrete measurable VLP antigen into tissue culture media followingtransient transfection of COS-1 or CHO cells. The inability of cellstransfected with these plasmids to secrete VLP antigen intotissue-culture media could result from the disruption of a variety ofprotein maturation processes. Without being bound by theory,interference with particle maturation could occur via disruption ofE-prM/M intermolecular interactions, E-glycoprotein dimer interactions,or via the disruption of dimer organization into the surface latticecovering mature particles. Although the two processes areinterdependent, these substitutions may not interfere with particleformation per se, but may directly interfere with particle secretionitself. In fact, the IFA staining pattern of DEN-2 G₁₀₄H and of W₂₃₁F/Ltransfected cells was highly punctate and localized within inclusionbodies. Similar IFA staining patterns have been observed withnon-secreting constructs of dengue and other flaviviruses (Chang et al.,Virology 306:170-80, 2003). Studies with TBE virus VLPs have shown thatinteractions between prM and E are involved in prM-mediatedintracellular transport of prM-E heterodimers (Allison et al., J. Virol.73:5605-12, 1999). The location of Gly₁₀₄ near the interior-lateral edgeof DII puts it very close to the E-dimer “hole” where the prM/M proteinsare located in the heterodimer (Kuhn et al., Cell 108:717-25, 2002; FIG.1). Therefore, it seems likely that G₁₀₄H interferes with VLP secretionvia disruption of the prM-E interactions necessary for intracellulartransport and secretion. The identity of this residue is positivelycorrelated with arthropod vector. The mosquito-born flaviviruses have aglycine at this position whereas the tick-borne flaviviruses have ahistidine. Interestingly, Pletnev et al. (J. Virol. 67:4956-63, 1993)introduced the reverse substitution, H₁₀₄G, into the TBE virusE-glycoprotein in a TBE/DEN-4 chimeric infectious clone, and they wereunable to recover virus from this mutant. The inability of G₁₀₄Htransfected cells to secrete VLP antigen similarly suggests that thistoo could be a lethal substitution in DEN-2 virus. Taken together, thesetwo results are consistent with the idea that vector-specific selectionhas produced strong epistasis between this residue and otherunidentified residue(s) elsewhere in the E- or prM/M proteins.

EXAMPLE 4 Identification of Flavivirus Complex and Sub-ComplexCross-Reactive Epitope Residues

This example demonstrates the identification of flavivirus complex andsub-complex cross-reactive epitopes using a structure-based rationalmutagenesis method.

Cell Culture, Virus Strains and Recombinant Plasmids

CHO cells (ATCC CCL 61; Manassas, Va.) were grown at 37° C. with 5% CO₂on Dulbeco's modified Eagle's minimal essential medium with F-12nutrient mixture (D-MEM/F-12, GIBCO, Grand Island, N.Y.) supplementedwith 10% heat-inactivated fetal bovine serum (FBS, Hyclone Laboratories,Inc., Logan, Utah), 110 mg/l sodium pyruvate, 0.1 mM nonessential aminoacids, 2 mM L-glutamine, 2.438 g/L NaHCO₃, 100 U/ml penicillin, and 100μg/mil streptomycin.

The recombinant expression plasmids pCB8SJ2 and pCBWN were used astemplate DNAs for both site-directed mutagenesis and for transientexpression of St. Louis encephalitis virus (SLEV) and West Nile virus(WNV) recombinant antigen (see below). The pCB8SJ2 plasmid includes thehuman cytomegalovirus early gene promoter, Japanese encephalitis virus(JEV) signal sequence, SLEV prM and E gene region (amino-terminal 80%),JEV carboxyl terminal 20%, and bovine growth hormone poly(A) signal. Thereplacement of the terminal 20% of SLEV E with JEV E gene sequencesdramatically increases the secretion of extracellular VLPs into theculture medium without altering the native SLEV E glycoproteinconformation (Purdy et al., J. Clin. Micro. 42:4709-17, 2004). The pCBWNplasmid includes the human cytomegalovirus early gene promoter, JEVsignal sequence, WNV prM and E gene region in its entirety, and bovinegrowth hormone poly(A) signal (Davis et al., J. Virol. 75:4040-47,2001).

Procedural Algorithm

Following the identification and ablation of flavivirus groupcross-reactive epitopes, flavivirus complex and sub-complexcross-reactive epitopes have been identified. Two different flaviviruscomplexes, the JEV complex and the DENV complex, were focused on. TheDENV complex consists of the four dengue serotypes, DENV-1, DENV-2,DENV-3, and DENV-4. The large JEV complex includes JEV, WNV, MurrayValley encephalitis virus (MVEV), and SLEV.

The procedural algorithm for the identification of flavivirus complexand sub-complex cross-reactive epitopes utilizes the followingoptimality criteria: 1) The identification and selection of amino acidresidues with ≧35% of their surface solvent accessible. These residuesare identified from the published atomic structure coordinates of theDENV-2 soluble ectodomain of the envelope glycoprotein and homologymodels of SLEV and WNV derived from the DENV-2 structure (Modis et al.,Proc. Natl. Acad. Sci. USA 100:6986-91, 2003). In addition toexamination of amino acid residues in structural domain II, residues indomains I and III were examined, since published results indicate thatsome complex and sub-complex cross-reactive epitopes are mapped ontodomains I and III in addition to domain II (Roehrig et al., Virology246:317-28, 1998). 2) Amino acids on the outer or lateral surface of theE-glycoprotein dimer, and accessible to antibody. 3) Amino acidconservation across the flavivirus complex (based upon a structuralalignment of the protein sequences). Residues conserved across allmember viruses of the same complex are favored. If conserved within butnot across the entire complex, then residues with shared identitiesbetween WNV and SLEV are favored in the JEV complex, and residues withshared identities between DENV-2 and two or more other viruses in theDENV complex are favored over those shared with DENV-2 and only oneother DENV complex virus. 4) Side chain projections exposed towards theouter surface and accessible to antibody paratopes. 5) Residues withhigh temperature (β-) factors should be favored, as these residues tendto be flexible and are able to conform to the antibody paratope,increasing the antibody-antigen affinity. Amino acid residues with hightemperature factors are more commonly found in antigen epitopes thanlower temperature factor residues. 6) Following identification ofpotential individual flavivirus complex and sub-complex cross-reactiveepitope residues, all residues are mapped and highlighted on the sameE-glycoprotein dimer structure together. With this technique, groups ofpotential cross-reactive epitope residues forming clusters (and henceprobable epitopes) are readily identified. 7) Residues fitting all ofthese criteria and occurring in structural clusters approximately 20×30Å² (which is the average “footprint” of an antibody Fab that interactswith an antigen epitope) are favored over residues that are moreisolated in the protein structure. 8) Within an identified structuralcluster of potential epitope residues, residues that more completelysatisfy greater numbers of the optimality criteria are selected for thefirst round of mutagenesis analysis.

Site-Directed Mutagenesis

Site-specific mutations were introduced into the SLEV and WNV E genesusing the Stratagene Quick Change® multi site-directed mutagenesis kit(Stratagene, La Jolla, Calif.) and pCB8SJ2 and pCBWN as DNA templatesfollowing the manufacturer's recommended protocols. The sequences of themutagenic primers used for all constructs are listed in Table 5. Four orfive colonies from each mutagenic PCR transformation were selected andgrown in 5 ml LB broth cultures. DNA was mini-prepped and sequenced fromthese cultures. Structural gene regions and regulatory elements of allpurified plasmids were sequenced entirely upon identification of thecorrect mutation. Automated DNA sequencing was performed using a BeckmanCoulter CEQ™ 8000 genetic analysis system (Beckman Coulter, Fullerton,Calif.) and analyzed using Beckman Coulter CEQ™ 8000 (Beckman Coulter,Fullerton, Calif.) and Lasergene® software (DNASTAR, Madison, Wis.).

Transient Expression of SLEV and WNV Recombinant Antigens by CHO Cells

CHO cells were electroporated with pCB8SJ2 or pCBWN using the protocoldescribed by Chang et al. (J. Virol. 74:4244-52, 2000). Electroporatedcells were recovered in 50 ml DMEM, seeded into 150 cm² culture flasksfor VLP expression and into 96-well tissue culture plates for IFA, andincubated at 37° C. with 5% CO₂. Cells in 96 well plates for IFA werefixed 14-24 hours post electroporation. Tissue-culture medium and cellswere harvested 48-72 hours post electroporation for antigencharacterization.

Characterization of Mutant pCB8SJ2 and pCBWN Infected Cells and SecretedAntigen

Fourteen to twenty four hours following electroporation, 96-well tissueculture plates (Costar® #3603 Corning, Inc., Corning, N.Y.) containingcells transformed with the mutated pCB8SJ2 or pCBWN clones were washedtwice with PBS, fixed with 3:1 acetone:PBS (v:v) for 10 minutes and airdried. E-glycoprotein-specific mAbs recognizing each of the threeE-glycoprotein domains (Table 6) were used to determine affinityreductions in cross-reactive epitopes by IFA as described by Chang etal. (J. Virol. 74:4244-52, 2000).

Tissue culture medium was harvested 48-72 hours followingelectroporation. Cell debris was removed from tissue culture media bycentrifugation for 30 minutes at 10,000 rpm. Ag-ELISA was used to detectsecreted antigen from the mutagenized pCB8SJ2 and pCBWN transformed CHOcells. Secreted antigen was captured with polyclonal rabbit anti-SLEVand rabbit anti-pCBWN sera at 1:30,000 and 1:50,000 dilutions,respectively. MHIAF specific for SLEV and WNV was used at a 1:15,000dilution to detect captured antigen, and this MHIAF was detected usinghorseradish peroxidase conjugated goat anti-mouse HIAF at a 1:5000dilution.

Secreted antigen was concentrated from positive tissue culture medium bycentrifugation overnight at 19,000 rpm, and resuspended in TN buffer (50mM Tris, 100 mM NaCl, pH 7.5) to 1/100^(th) the original volume.Alternatively, some antigens were concentrated using Millipore's Amicon®Ultra PL-100 (Millipore, Billerica, Mass.) centrifugal filter devices.Concentrated antigen was analyzed with a panel of anti-flavivirus mAbsin Ag-ELISA to determine mAb end point reactivities of the mutatedantigens, following the protocol of Roehrig et al. (Virology 246:317-28,1998). This Ag-ELISA protocol is the same as that used herein to detectsecreted antigen, with the exception of using the specified mAbs (Table6) instead of polyclonal MHIAF.

Antigenic Characterization and MAb Screening of Potential Cross-ReactiveEpitope Residue Mutants

Using the structure-based design approach described above, candidateflavivirus complex and sub-complex cross-reactive epitope residues werenarrowed down to 34 in DENV-2 and 31 each in WNV and SLEV. From theseresidues and with reiterative application of the optimality criteriadescribed herein 17 DENV-2, 13 WNV, and 11 SLEV residues were chosen asmost likely to be incorporated into complex and sub-complexcross-reactive epitopes (highlighted in Tables 7-9). Amino acidsubstitutions were modeled at these probable cross-reactive epitoperesidues, selecting substitutions that should potentially disrupt orablate antibody recognition without altering E-glycoprotein structuralconformation, disrupting dimer interactions, or impairing particleformation, maturation, or secretion. Stability calculations wereperformed for all possible amino acid substitutions of candidateresidues using the PoPMuSiC server, (available on the Université Librede Bruxelles' web site) and the DENV-2 E-glycoprotein pdb filecoordinates (Modis et al., Proc. Natl. Acad. Sci. 100:6986-91, 2003) orhomology model coordinates for WNV and SLEV. Amino acid substitutionsmodeled in the E-glycoprotein structures with free energies of foldingequal to or less than that of the non-mutated wild-type E-glycoproteinwere re-examined with the Swiss-Pdb Viewer software (available on theSwiss Institute of Bioinformatics' web site) to identify thosesubstitutions that minimized local structural disturbances whilemaintaining structurally relevant biochemical interactions such ashydrogen bonding and/or charge interactions with neighboring aminoacids.

Substitutions at 11 of 16 potential cross-reactive epitope residuesselected for mutagenesis in pCB8SJ2 altered the reactivities of all 14of the anti-SLE mAbs, relative to wild-type pCB8SJ2 (Table 10). Eight ofthe 14 MAbs were flavivirus group- or subgroup-cross-reactive (see Table6). Substitutions at nine of the 16 residues analyzed altered thereactivity of all eight of the flavivirus group- orsubgroup-cross-reactive mAbs. Substitutions at four of 16 potentialcross-reactive epitope residues altered all three of the JEV complex-and subcomplex-cross reactive mAbs. Only one substitution however,affected type-specific mAb reactivities (FIG. 3). The effect of thissubstitution (G₁₀₆Q) on type-specific mAb reactivities was to actuallyincrease the reactivity of these mAbs relative to that of the wild-typeunaltered pCB8SJ2. Without being bound by a single theory, such increasein the reactivity of type-specific antibodies is believed to bebeneficial for the development of type-specific flavivirus antigens.

Substitutions at 14 of 17 residues selected for mutagenesis in pCBWNaltered the reactivities of all 10 of the anti-WNV mAbs, relative towild-type pCBWN (Table 11). Six of the 10 anti-WNV mAbs were flavivirusgroup- or subgroup-cross-reactive, two were JEV complex cross-reactiveand two were WNV type-specific (see Table 6). Nine of the 17substitutions examined altered the reactivities of all six group- andsubgroup-cross-reactive mAbs; 12 of these 17 substitutions affected thereactivities of both of the JEV complex cross-reactive mAbs. The G₁₀₆Vsubstitution in pCBWN was the only substitution to alter type-specificmAb reactivities, and, as with pCB8SJ2, this substitution actuallyincreased the reactivity of the type-specific mAbs (FIG. 3).

The outcome that many of these substitutions altered mAb reactivities(Tables 10 and 11; FIG. 3) illustrates not only the efficiency of thedescribed algorithms for identifying cross-reactive epitope residues,but also that these cross-reactive epitopes can be altered to ablate orappreciably interfere with the ability of an antibody to recognize thesemodified antigens. For example, 82% and 69% of the potentialcross-reactive epitope residue substitutions examined in pCBWN andpCB8SJ2, respectively, affected all of the cross-reactive antibodiesreactive to these two viruses from the antibody panel (see FIG. 3). Thehigh percentage of residues, selected a priori, affecting mAbreactivities illustrates the accuracy of the cross-reactive epitoperesidue selection algorithms.

The mAb characterization of potential cross-reactive epitope residuemutants illustrates the importance of the E-protein fusion peptideregion as a potently cross-reactive antigenic determinant. As describedherein, substitutions at fusion peptide residues G₁₀₄, G₁₀₆, and L₁₀₇strongly affected many of the mAb reactivities for DENV-2, SLEV and WNV(see Tables 10 and 11). Without being bound by a single theory, G₁₀₆appears to be the most important cross-reactive antigenic determinant ofthese residues. Substitutions at G₁₀₆ altered the reactivities of 7 of10 cross-reactive mAbs recognizing SLEV, and 7 of 8 cross-reactive mAbsrecognizing WNV (see Tables 10 and 11). Substitutions at fusion peptideresidue G₁₀₄ also affected the reactivities of many mAbs for each ofthese viruses. However, all substitutions examined at this positionproduced plasmids that were unable to efficiently secrete VLP antigenupon transient transformation into eukaryotic cells. This observationwas true for all three flaviviruses examined: DENV-2, SLEV and WNV.

Substitutions at fusion peptide residue G₁₀₆ had a variety of effects onmAb reactivities for both pCBWN and pCB8SJ2. The majority of thesubstitutions at this residue reduced or ablated a mAb's ability torecognize the antigen. This occurred with cross-reactive mAbs 4G2,6B6C-1, 4A1B-9, and 2B5B-3 in G₁₀₆V-pCBWN and with 4G2 and 2B5B-3 forG₁₀₆Q-pCB8SJ2 (see Tables 10 and 11), indicating that the substitutedresidue is a part of the antigenic epitope recognized by theseantibodies.

EXAMPLE 5 Human IgM MAC-ELISA Serology

This example demonstrates the representative nature of a murine antibodyresponse as a model of human antibody response to substitutions in theflavivirus cross-reactive epitopes.

Human Sera

Well-characterized serum specimens were assembled from the Diagnosticand Reference Laboratory, Arbovirus Diseases Branch, Division ofVector-Borne Infectious Diseases, US Centers for Disease Control andPrevention. A serum panel (see Table 12) was assembled from patientsinfected in the US between 1999 and 2004 with either WNV (n=6) or SLEV(n=10), as determined by the standard 90% plaque-reductionneutralization (PRNT) assay. SLEV is endemic to North America, whereasWNV was first introduced into North America in 1999 and has spreadepidemically since that time.

The flavivirus responsible for the most recent infection was determinedas that with the highest neutralizing antibody titer, which had to be atleast four-fold greater than that for any other virus tested. Because ofthe high level of cross-reactivity between the SLEV and WNV viruses, itis often difficult to determine the infecting virus by ELISA, thusrequiring the PRNT. SLEV infected sera with measurably high levels ofcross-reactivity for WNV were purposefully selected in order to maximizethe ability to assess for improved discrepancy (specificity) of thepCBWN-G₁₀₆V versus the pCBWN wild-type antigen. SLEV infected patientsera were split into two groups based upon previously determined(Diagnostic and Reference Laboratory) positive to negative (P/N) ratiosfor SLEV and for WNV. ‘Equivocal’ SLEV sera (n=5) were those that wereclear SLEV infections from the PRNT data, yet had MAC-ELISA P/N ratiosthat were not statistically different between SLEV and WNV. Three ofthese equivocal SLEV samples were negative (P/N≦2.0) for both viruses,one was presumptive positive (P/N≧2.0 and <3.0), and one was definitivepositive (P/N>5.0) for both viruses. ‘Misleading’ SLEV sera (n=5) wereSLEV positive in the PRNT, yet had MAC-ELISA P/N ratios that were notonly positive for both viruses, but were actually greater for WNV thanfor SLEV. Definitive ‘positive’ WNV infected patient sera (n=6) wereselected based on MAC-ELISA results from the Diagnostic and ReferenceLaboratory collection for use as positive control sera to assess theaccuracy of the pCBWN-G₁₀₆V plasmid derived antigen.

IgM ELISA Protocols

IgM ELISAs were performed following the protocols of Purdy et al. (J.Clin. Micro. 42:4709-17, 2004) and Holmes et al. (J. Clin. Micro.43:3227-36, 2005). Briefly, the inner 60 wells of Immulon II HBflat-bottomed 96-well plates (Dynatech Industries Inc., Chantilly, Va.)were coated overnight at 4° C. in a humidified chamber with 75 μl ofgoat anti-human IgM (Kierkegaard & Perry Laboratories, Gaithersburg,Md.) diluted at 1:2000 in coating buffer (0.015 M sodium carbonate,0.035 M sodium bicarbonate, pH 9.6). Wells were blocked with 300 μl ofInBlock blocking buffer (Inbios, Seattle, Wash., L/N FA1032) for 60minutes at 37° C. in a humidified chamber. 50 μl of sera were added toeach well and incubated again for 60 minutes at 37° C. in a humidifiedchamber. Human test sera were diluted 1:400 in sample dilution buffer(Inbios, L/N FA1055). Positive control sera were diluted 1:3000 for SLEVand 1:800 for WNV. Positive and negative control VLP antigens weretested on all patient sera in triplicate by diluting appropriately insample dilution buffer and adding 50 μl to appropriate wells forincubation overnight at 4° C. in a humidified chamber. Captured antigenswere detected with 50 μl/well of polyclonal rabbit anti-pCBWN diluted1:1000 in sample dilution buffer and incubated for 60 m at 37° C. in ahumidified chamber. Rabbit sera was detected with horseradish peroxidaseconjugated goat anti-rabbit sera diluted 1:8000 in IgM conjugatedilution buffer (Inbios, L/N FA1056) and incubated for 60 m at 37° C. ina humidified chamber. Bound conjugate was detected with 75 μl of3,3′5,5′-tetramethylbenzidine (Neogen Corp, Lexington, Ky.) substrate,incubated at RT for 10 min, stopped with 50 μl of 2NH₂SO₄, and then readat A₄₅₀ using an ELx405HT Bio-Kinetics microplate reader (Bio-TekInstruments Inc., Winooski, Vt.).

IgM test Validation and Interpretation

Test validation and P/N values were determined according to theprocedure of Martin et al. (J. Clin. Micro. 38:1823-26, 2000), usinginternal positive and negative serum controls included in each 96-wellplate. Positive (P) values for each specimen were determined as theaverage A450 for the patient serum sample incubated with positive VLPantigen. Negative (N) values were determined for each plate as theaverage A₄₅₀ for the normal human serum control incubated with positiveVLP antigen.

Human Serology

To determine how representative the murine antibody response (mAb data)is as a model of the human antibody response (serological data) to theviral substitution antigens described herein, serological assays wereperformed with single substitution, prototype type-specific antigens. Asthe mAb screening results indicated that fusion peptide residue 106 wasincorporated into multiple cross-reactive epitopes for both WNV andSLEV, this substitution was selected to conduct MAC-ELISA serum tests.

The prototype type-specific G₁₀₆V-WNV Ag dramatically outperformed thewild-type (wt)-WNV Ag when tested on 10 difficult to discern ‘equivocal’or positively ‘misleading’ SLEV-infected patient sera (Table 12). Six of10 of these SLEV infected sera were correctly diagnosed as WNV-negativeby MAC-ELISA (P/N≦2.0) with the G₁₀₆V-WNV prototype Ag, three were‘equivocal’ (P/N>2.0≦3.0) and one was WNV positive. However, when thesesame sera were tested with the wt-WNV Ag, only four sera were correctlyscored as WNV negative, one was equivocal, and five were misdiagnosed asWNV positive with this unmodified Ag. When antigens were directlycompared on each individual serum sample, the G₁₀₆V-WNV Ag producedlower P/N ratios than did the wt-WNV Ag in nine of 10 cases on theseSLEV infected sera, indicating that the G₁₀₆V-WNV Ag exhibits improvedspecificity and reduced cross-reactivity relative to the wt-WNV Ag.

The prototype type-specific G₁₀₆V-WNV Ag also outperformed the unalteredwt-WNV Ag in MAC-ELISA sensitivity tests on positive WNV infected humansera (Table 12). Five of six WNV infected patient sera had positive P/Nratios when tested with the G₁₀₆V-WNV Ag, whereas four were positivewith the wt-WNV Ag. The single WNV positive serum sample that testednegative with the wt-Ag and equivocal with the G₁₀₆V Ag had the lowestneutralizing titers of the WNV sera in the PRNT (see Table 12),indicative of a weak antibody titer.

In addition to improved accuracy with the G₁₀₆V-WNV Ag, it was also moresensitive than was the wt-WNV Ag. In 5 of the 6 WNV infected sera, theMAC-ELISA P/N ratios were higher with the G₁₀₆V- than with the wt-WNV Ag(Table 12). Higher P/N ratios are expected from an improvedtype-specific Ag relative to the cross-reactive wt Ag when tested onsera infected with the same virus.

The prototype type-specific G₁₀₆V-WNV Ag exhibited improved specificity,accuracy, and sensitivity relative to the unmodified wt-WNV Ag. TheG₁₀₆V-WNV Ag was more specific and accurate for WNV diagnosis than wasthe wt Ag, correctly diagnosing more WNV infected sera as positive andfewer SLEV infected sera as negative, than did the wt-WNV Ag. TheG₁₀₆V-WNV Ag was also more sensitive at detecting WNV antibody in WNVinfected serum than was the wt-WNV Ag. The positive signal indicatingthe presence of WNV antibody (P/N ratios) was greater for G₁₀₆V-WNV Agthan it was for the wt-Ag when testing WNV infected sera, and less thanthat of the wt-Ag when testing non-WNV infected sera.

EXAMPLE 6 Murine Immunization

This example demonstrates the ability of prototypical type-specificflavivirus mutant compositions to generate type-specific neutralizingantibody responses in mice.

Mouse Vaccination

Groups of six female outbred ICR mice were used in this study. Mice wereimmunized by injection with pCB8D2-2J-2-9-1, pCB8D2-2J-2-9-1-G₁₀₆Q,pCBWN, pCBWN-G₁₀₆V, pCB8SJ2, or pCB8SJ2-G₁₀₆Q expression plasmids asdescribed herein. Each mouse was injected with 100 μg of Picogreen®fluorometer quantified plasmid DNA in PBS pH 7.5, at a concentration of1 μg/μl Mice were immunized with 50 μg of plasmid DNA injectedintramuscularly into each thigh on weeks 0 and 3. Mice were bled on weeksix following initial vaccination.

Plaque Reduction Neutralization Assays

Six week post-vaccination serum specimens were tested for the presenceof type-specific neutralizing (Nt) antibody (Ab) by plaque reductionneutralization test (PRNT). PRNT was performed with freshly confluentVero cell monolayers as described by Chang et al. (J. Virol. 74:4244-52,2000) using DENV-2 (16681), WNV (NY-99), and SLEV (MSI-7) viruses.

Neutralizing Antibody Responses

Mice were immunized with wild-type and G₁₀₆ substituted plasmids forWNV, SLEV, and DENV-2 to determine if there were differences between thewild-type and G₁₀₆ prototype type-specific antigens for type-specific NtAb titer, cross-reactive Nt Ab titer, and protection from viruschallenge. The type-specific Nt Ab titer results are shown in Table 13.There was little difference in the 75% PRNT titer between wt and G₁₀₆substituted plasmids for all three viruses. The 75% Nt Ab titer wasgreater than or equal to 1:128 for almost all of the mice immunized withboth the DENV-2 and both the WNV DNA vaccines. One mouse immunized withthe wt DENV-2 DNA vaccine had a 75% PRNT titer of 1:64, and two miceimmunized with the pCBWN-G₁₀₆V DNA vaccine had 75% PRNT titers of 1:64and 1:16.

These results demonstrate that for all three flaviviruses tested, therewas little to no detectable difference in type-specific neutralizingantibody titer between the prototype type-specific G₁₀₆ mutant vaccinesand their wt counterparts. These results also illustrate that themethods described herein for ablating cross-reactive epitope residuescan be used to generate type-specific flavivirus prM/E expressionplasmids for use as DNA vaccines that still maintain potenttype-specific neutralizing immunogenicity.

EXAMPLE 7 Reduction of Cross-Reactive Immunogenicity of Type-SpecificGenetic Vaccines

This example provides methods by which prototypical type-specificflavivirus mutant compositions can be used to generate a reducedcross-reactive neutralizing antibody response relative to the unalteredwild-type compositions.

Mouse Vaccination and Plaque Reduction Neutralization Assays

Female outbred ICR mice (such as the mice in Example 6) can be used inthis study. Twelve-week post vaccination serum samples from immunizedmice will be tested for cross-reactive (heterologous) Nt antibodyresponse by PRNT. Unlike the type-specific PRNTs performed in Example 6,the cross-reactive PRNTs will be performed by examining Nt of immunizedmouse sera not only for the type-specific virus used for immunization,but also for Nt of the seven other medically important flaviviruses.Thus, all 12-week mouse sera will be tested for neutralization againsteight different flaviviruses: all four dengue serocomplex viruses,DENV-1 (16007), DENV-2 (16681), DENV-3 (H87), and DENV-4 (H241); threeJEV serocomplex viruses, JEV (SA14-14-2), WNV (NY-99) and SLEV (MSI-7);and the single medically important member of the yellow fever virusserocomplex, YFV (17D).

Predicted Antibody Response

Without being bound by theory, similar type-specific Nt Ab titersbetween the prototype type-specific G₁₀₆ mutant vaccines and their wtcounterparts are expected. Thus, both pCBWN and pCBWN-G₁₀₆V vaccinatedmouse sera are predicted to have similar Nt Ab titers against WNV, andpCB8D2-2J-2-9-1 and pCB8D2-2J-2-9-1-G₁₀₆Q will have similar Nt Ab titersagainst DENV-2. However, when these same sera are tested for Nt againstthe heterologous flaviviruses, it is expected that significantly lowerPRNT titers for prototype type-specific G₁₀₆ mutant vaccinated mousesera will be observed than for the counterpart wt vaccinated mouse sera.For example, mice immunized with pCBWN and pCBWN-G₁₀₆V will both havesimilar PRNT titers against WNV, whereas, pCBWN-G₁₀₆V immunized micewill have significantly lower PRNT titer against SLEV, JEV, YF, and thefour dengue serotype viruses, than wild-type pCBWN immunized mice.

EXAMPLE 8 Combining Multiple Cross-reactive Epitope Substitutions intoSingle Plasmid Constructs

This example provides methods by which individual substitutionsaffecting different flavivirus cross-reactive epitopes can be combinedinto a single construct.

Individual substitutions affecting different flavivirus cross-reactiveepitopes (such as those disclosed herein) can be combined into a singleconstruct based, for example, on mAb screening results disclosed herein(see, Tables 3, 10 and 11), as well as additional mAb screening studies.For example, a mutagenesis primer has been designed for SLEV tointroduce both the G₁₀₆Q and L₁₀₇K substitutions into a single pCB8SJ2plasmid (see, Table 5). This double mutation plasmid has beenconstructed, and its sequence confirmed.

Cells can be transformed with this double mutated plasmid (or anotherplasmid containing a sequence encoding an E glycoprotein having acombination of two or more mutated amino acids), and the antigencharacterized. In SLEV, the G₁₀₆Q substitution alone alters thereactivities of many mAbs recognizing distinct cross-reactive epitopes(Table 10). However, this substitution alone has no significant effecton the flavivirus group cross-reactive epitope recognized by MAb T-23-1.The L₁₀₇K substitution does knock out the ability of mAb T-23-1 torecognize the flavivirus cross-reactive epitope. Without being bound bytheory, this suggests that L₁₀₇ is incorporated in the cross-reactiveepitope recognized by mAb T-23-1, while G₁₀₆ is not.

Because of the generally additive effects observed when combining thesesubstitutions into single constructs (see, Tables 10 and 11), it isexpected that G₁₀₆Q/L₁₀₇K antigen will combine the different effectsobserved from mAb screening of the individual mutants into a single,multiple substituted mutant. Upon transfection into mammalian cells,such a multiple mutant plasmid can be used to produce improvedtype-specific antigens. When utilized as genetic vaccines, theseplasmids are expected to exhibit further reductions in cross-reactiveimmunogenicity while still inducing a potent type-specific immuneresponse.

EXAMPLE 9 Immune Stimulatory Compositions for the Inhibition orTreatment of a Flavivirus Infection

This example provides methods for administering substances suitable foruse as immune stimulatory compositions for the inhibition or treatmentof a flavivirus infection.

An immune stimulatory composition containing a therapeutically effectiveamount of a flavivirus polypeptide that includes at least one flaviviruscross-reactive epitope with reduced or ablated cross-reactivity(particularly in an E glycoprotein) can be administered to a subject atrisk for, or exposed, to a flavivirus (e.g., a dengue virus, West Nilevirus, etc.). Alternatively, an immune stimulatory compositioncontaining a therapeutically effective amount of a nucleic acid vectorthat includes flavivirus nucleic acid molecules described herein, orthat includes a nucleic acid sequence encoding at least one flaviviruscross-reactive epitope with reduced or ablated cross-reactivity(particularly in an E glycoprotein), can be administered to a subject atrisk for, or exposed to a flavivirus.

Dosages and routes of administration for the immune stimulatorycomposition can be readily determined by one of ordinary skill in theart. Therapeutically effective amounts of an immune stimulatorycomposition can be determined, in one example, by in vitro assays oranimal studies. When in vitro or animal assays are used, a dosage isadministered to provide a target tissue concentration similar to thatwhich has been shown to be effective in the in vitro or animal assays.

While this disclosure has been described with an emphasis on preferredembodiments, it will be apparent to those of ordinary skill in the artthat variations and equivalents of the preferred embodiments may be usedand it is intended that the disclosure may be practiced otherwise thanas specifically described herein. Accordingly, this disclosure includesall modifications encompassed within the spirit and scope of thedisclosure as defined by the claims below.

APPENDIX I Tables

TABLE 1 Nucleotide sequence of primers used for mutagenesis. SEQ IDSequence NO: Mutation 5′-TGTTGTTGTGTTGGTTAGGTTTGCCTCTATACAG- 1 K₆₄N 3′5′-TGGGTTCCCCTTGCATTGGGCAGCGAGATTCTGTTG 2 T₇₆M TTG-3′5′-TTCATTTAGGCTGGGTTCCCCTCGTGTTGGGCAG-3′ 3 Q₇₇R5′-CCCTTTCCAAATAGTCCACAGTGATTTCCCCATCCT 4 G₁₀₄H CTGTCTACC-3′5′-GCCTCCCTTTCCAAATAGTTGACATCCATTTCCCC 5 G₁₀₆Q A-3′5′-GGTCACAATGCCTCCCTTTCCAAATTTTCCACATCC 6 L₁₀₇K ATTTCCCC-3′5′-AGTTTTCTGGTTGCACAACTTTTCCTGCCATGTTCT 7 E₁₂₆A TTTTGC-3′5′-GTATCCAATTTGACCCTTGATTGTCCGCTCCGGGCA 8 T₂₂₆N ACC-3′5′-GTCTCTTTCTGTATGAAATTTGACCCTTGTGTGTC- 9 W₂₃₁F 3′5′-AATGTCTCTTTCTGTATCAGATTTGACCCTTGTGTG 10 W₂₃₁L TCCGCTCC-3′5′-TCCTGTTTCTTCGCACGGGGATTTTTGAAAGTGACC- 11 H₂₄₄R 3′5′-ACAACAACATCCTGTCGCTTCGCATGGGGATTTTTG- 12 K₂₄₇R 3′ The mismatchednucleotides causing the desired substitutions are underlined.

TABLE 2 Stability free energy (ddG) calculations for putative domain IIcross-reactive epitope substitutions based upon the published pdbcoordinates for the DEN-2 virus (Modis et al., PNAS 100: 6986-91, 2003)and the TBE virus (Rey et al, Nature 375: 291-98, 1995) E-glycoproteinstructures. DEN-2 SUB ddG (kcal/mol) TBE SUB ddG (kcal/mol) K64N −0.45K64N −0.15 T76M −0.54 T76M −0.02 Q77R 0.45 M77R −0.10 G104H −0.16 H104HNA G106Q 0.87 G106Q −0.03 L107K 0.19 L107K 0.12 E126A 2.16 K126A 0.85T226N 0.33 Q233N 0.03 E126A/T226N 2.49 K126A/Q233N 0.88 W231F 1.54 W235F1.34 W231L 1.84 W235L 2.26 H244R 4.18 H248R 0.00 K247R −0.30 K251R −0.19

TABLE 3 mAb reactivities for mutant and wild-type plasmids. mAb D2HIAF4G2 6B6C1 4E5 1A5D1 1B7-5 10A1D2 Epitope polyclonal A1 A1 A2 A3 A5 A/CPRNT + + +/− + − + − SA + + + − +/− + +/− Specificity NA group groupsub-comp. type sub-group comp. Wild Type IFA 4.1 3.8 3.8 2.6 4.4 4.1≧2.9 Ag-ELISA >6.0 >6.0  ≧6.0  ≧2.9 4.2 5.7 >3.8 T76M IFA — — — — — — —Ag-ELISA — — — — ≦5% — — G104H IFA — <3% 6% — <0.8%    3% — Ag-ELISA nana na na na na na G106Q IFA — <3% <3%  — <0.8%   — nd Ag-ELISA — <0.1%  <0.1%   — <6% —  13% L107K IFA — <3% — — — — <25% Ag-ELISA — <0.1%   — — 5% —  6% E126A IFA — — 6% — — — — Ag-ELISA — — — 10% 13% — E126A/T226NIFA — — 3% — —  3% <25% Ag-ELISA — — —  5% 13% — W231F/L IFA — <3% <3% — — <2% — Ag-ELISA na na na na na na — mAb 1B4C2 9A4D1 3H5 9A3D8 10A4D29D12 Epitope C1 C4 B1 B2 B3 B4 PRNT − − + + + + SA + − + + + +Specificity sub-comp. type type type sub-comp. type Wild Type IFA 4.4≧2.9 >4.4 3.5 4.1 >4.4 Ag-ELISA >5.3  2.9 >6.0 >6.0 >6.0 >6.0 T76M IFA —— — nd — nd Ag-ELISA 0.8%   — — — — — G104H IFA — — — nd — nd Ag-ELISAna na na na na na G106Q IFA 6% nd — — — — Ag-ELISA ≦0.1%   — — — — —L107K IFA 6% — — nd — nd Ag-ELISA 0.2%   — — — — — E126A IFA — — — nd —nd Ag-ELISA — — — — — — E126A/T226N IFA — — — nd — nd Ag-ELISA — — — — —— W231F/L IFA 6% — — — — — Ag-ELISA — — — — — — na: not applicable(these constructs did not secrete VLP antigen and thus could not beexamined by Ag-ELISA); nd: not determined.

TABLE 4 Amino acid sequence variability for proposed cross-reactiveepitope residues in domain II of the flavivirus E protein. Virus K64NT76M Q77R G104H G106Q L107K E126A T226N W231/F, L H244R K247R DEN-2 K TQ G G L E T W H K DEN-4 S T Q G G L T T W H R DEN-3 K T Q G G L E T W HK DEN-1 K T Q G G L E T W H K Japanese Encephalitis S T T G G L I T W HK Murray Valley encephalitis T T T G G L A T W H K West Nile T T M G G LI T W H K St. Louis encephalitis T T T G G L T T W H K Ilhéus T T M G GL T E W H R Rocio T T M G G L M D W H R Bagaza K T M G G L E G W H KIguape E Q M G G L P G W H K Bussuquara K A V G G L A S W H K Kokobera QT M G G L E G W H K Kédougou T T Q G G L K A W H K Zika S T Q G G L T TW H R Yellow fever V S T G G L S G W H T Sepik S T M G G L E G W H TEntebbe Bat N T T G G L Q D W H S Tick-borne encephalitis K T M H G L TQ W H K Louping ill K T M H G L T P W H K Omske hemorrhagic fever K A MH G L T V W H K Langat K T M H G L T E W H K Alkhurma K A M H G L T H WH K Deer tick K T T H G F V Q W H K Powassan K T T H G F V Q W H KMontana myotis D T L G G L A H W H K leukoencephalitis Rio Bravo S T Q GG L I S W H K Modoc E T Q G A L M P W Y K Apoi A T Q G G L I K W H KDENV-2 strains containing variable amino acid sequences at thesepositions are indicated below with their GenBank accession numbers (allincorporated by reference as of the date of filing of this application).64R: AF359579; 77L: M24449, X15434, X15214; 107F: M24446 126K: L10053,D00346, M29095, AF204178, M24450, M24451, AF410348, AF410361, AF410362,AF410365, AF204177, D10514 226K: AB111452, AY158337; 247R: AF231718,AF231719, AF231720

TABLE 5 Nucleotide sequence of primers used for mutagenesis. SEQ IDPrimer Sequence NO: Mutation SLEV: G104HCTCCCTTTTCCAAACAGACCACAGTGGTTACCCCATCCGC 31 Gly-His G104NCTCCCTTTTCCAAACAGACCACAGTTGTTACCCCATCCGC 32 Gly-Asn G104DCCCTTTTCCAAACAGACCACAGTCGTTACCCCATCCGC 33 Gly-Asp G104KCTCCCTTTTCCAAACAGACCACACTTGTTACCCCATCCGC 34 Gly-Lys G106QCTCCCTTTTCCAAACAGCTGACATCCGTTACCCCATCCGC 35 Gly-Gln G106KCTCCCTTTTCCAAACAGCTTACATCCGTTACCCCATCCGC 36 Gly-Lys G106VTCCCTTTTCCAAACAGTACACATCCGTTACCCCATCCGC 37 Gly-Val G106D CTTTTCCAAACAGATCACATCCGTTACCCCATCCGC 38 Gly-Asp L107FCTCCCTTTTCCAAAGAAACCACATCCGTTACCCCATCCGC 39 Leu-Phe G106Q/AATGCTCCCTTTTCCAAAGAACTGACATCCGTTACCCCATCCGC 40 Gly-Gln L107F Leu-PheR166Q CGGGCTTATGGTGAATTGAGCCGCTTGGTTTTTTCC 41 Arg-Gln T177ITTCCATACTCGCCCATGTTGGCAATAAAGGACGGTG 42 Thr-Ile G181SGTAACTGTTCCATACTCGGACATGTTGGCCGTAAAGG 43 Gly-Ser E182NGTAACTGTTCCATAGTTGCCCATGTTGGCCGTAAAGG 44 Glu-Asn T231NCTCTGTTGCGCCAATCGTTTGTGGCAGGGCTCGTC 45 Thr-Asn W233FTTCTCTGTTGCGGAAATCAGTTGTGGCAGGGCTCGTC 46 Trp-Phe H246RTACTACAGTTTGCTTGGTGGCACGCGGTTCCTC 47 His-Trp S276GTGATTGCAAGGTTAGGGTTGATCCGCTAACAGTGGC 48 Ser-Gly K294YCGTTCCCTTGATTTTGACGTAGTCAAGCTTAGCTCTGC 49 Lys-Tyr T301AACACATGCCATATGCCGTTCCCTTGATTTTGACC 50 Thr-Ala T330DCAGGGTCCGTTGCTTCCATCATACTGCAGTTCCAC 51 Thr-Asp A367SCGATCATGACCTTGTTGTTCGATCCCCCTGTGC 52 Ala-Ser N368FTTCGATCATGACCTTGTTGAACGCTCCCCCTGTGC 53 Asn-Phe WNV: G104NTTTGCCAAATAGTCCGCAGTTGTTGCCCCAGCCCC 54 Gly-Asn G104DTTGCCAAATAGTCCGCAGTCGTTGCCCCAGC 55 Gly-Asp G104KCCTTTGCCAAATAGTCCGCACTTGTTGCCCCAGCCCC 56 Gly-Lys G104ATTGCCAAATAGTCCGCATGCGTTGCCCCAGC 57 Gly-Ala G106VTTTGCCAAATAGGACGCAGCCGTTGCCCCAGCC 58 Gly-Val G106RTTTGCCAAATAGCCTGCAGCCGTTGCCCCAGCC 59 Gly-Arg G106YCCTTTGCCAAATAGGTAGCAGCCGTTGCCCCAGCCCC 60 Gly-Tyr G106ATTTGCCAAATAGAGCGCAGCCGTTGCCCCAGCC 61 Gly-Ala L107YTTCCTTTGCCAAAGTATCCGCAGCCGTTGCCCCAGCC 62 Gly-Tyr L107FCCTTTGCCAAAGAATCCGCAGCCGTTGCCCCAGC 63 Gly-Phe L107HCCTTTGCCAAAATGTCCGCAGCCGTTGCCCCAGC 64 Gly-His L107RCCTTTGCCAAATCTTCCGCAGCCGTTGCCCCAGC 65 Gly-Arg K118VCTTGGTAGAGCAGGCAAATACGGCGCATGTGTC 66 Lys-Val N154DCCAACCTGTGTGGAGTAGTCTCCGTGCGAC 67 Asn-Asp Y155GCCAACCTGTGTGGAGCCGTTTCCGTGCGACTC 68 Tyr-Gly Q158DCTGAGTGGCTCCAACATCTGTGGAGTAGTTTCCGTGCG 69 Gln-Asp R166YAGGAGTGATGCTGAAGTACCCTGCCTGAGTGG 70 Arg-Tyr T177VCCAAGCTTTAGTACGTATGAAGGCGCCGCAGGAG 71 Thr-Val E182GCCTCTCCATAGCCTCCAAGCTTTAGTGTGTATGAAGG 72 Glu-Gly W233FAACGTCTCTCTGTTCCTGAACACAGTACTTCCAGCAC 73 Trp-Phe S276DCCGACGTCAACTTGACAGTGTTGTCTGAAAATTCCACAGG 74 Ser-Asp K294NGTTCCCTTCAACTGCAAGTTTTCCATCTTCACTCTACAC 75 Lys-Asn T301NACAGACGCCATAGTTTGTTCCCTTCAACTGCAATTTTTCC 76 Thr-Asn T330NCCATCCGTGCCGTTGTACTGCAATTCCAACACCACAG 77 Thr-Asn A367VGGACCTTAGCGTTGACCGTGGCCACTGAAAC 78 Ala-Val N368SACCTTAGCGCTGGCCGTGGCCACTGAAAC 79 Asn-Ser The mismatched nucleotidescausing the desired substitutions are underlined.

TABLE 6 E-glycoprotein-specific mAbs recognizing each of the threeE-glycoprotein domains mAb Name Virus Specificity Domain 4G2 DENV-2group DII 6B6C-1 SLEV group DII T23-1 WNV group DII T23-2 JEV group DII2B6B-2 SLEV sub-grp (not WNV) DII 4A1B-9 MVEV group DII 1B7-5 DENV-3sub-grp: DEN + JE comp T21 DENV-3 sub-grp: DEN + JE comp 2B5B-3 SLEVsub-grp. JE comp + YF T11 DENV-3 sub-grp: DEN-2, 3, 4 + JE T5-1 JEVsub-grp: DEN-2, JE, SLE T5-2 JEV sub-grp: DEN-1, 2, JE, WN* 10A1D-2DENV-2 sub-grp: DEN-1, 2, 3, 4 + SLE DI/DII 6B4A-10 JEV JE comp. T16 JEVJE comp. 1B4C-2 DENV-2 sub-comp: DEN-2, 3 DI 10A4D-2 DENV-2 sub-comp:DEN-1, 2, 3 DIII 1B5D-1 SLEV sub-comp: SLE + JE E-2 T20 DENV-2 sub-comp:DEN-2 + JEV 4E5 DENV-2 sub-comp: DEN-1, 2, 3 DII 3H5 DENV-2 type DIII9A3D-8 DENV-2 type DIII 9D12 DENV-2 type DIII 1A5D-1 DENV-2 type DII9A4D-1 DENV-2 type DI T8 WNV type WNV 3.91D (KUNV) type WNV 3.67G (KUNV)type 4A4C-4 SLEV type 6B5A-2 SLEV type 1B2C-5 SLEV type

TABLE 7 Potential DENV-2 complex- and sub-complex-cross-reactive epitoperesidues, with residues chosen for mutagenesis highlighted

*not identified as ≧ 35% SA in this particular structure/model B-f:β-factor (temperature factor) a qualitative assessment of the scale(5-60Å²). SC?: is the amino acid side chain accessible and available forantibody binding Ep?: might this amino acid be incorporated into anantigen epitope? DVc: DENV1-4 complex; Jec: JE complex (medicallyimportant clade = JE, MVE, WN, SLE) SDM: site-directed mutagenesis

TABLE 8 Potential JEV complex- and sub-complex cross-reactive epitoperesidues from WNV, with residues chosen for mutagenesis highlighted

*not identified as ≧ 35% SA in this particular structure/model B-f:β-factor (temperature factor) a qualitative assessment of the scale(5-60Å²). SC?: is the amino acid side chain accessible and available forantibody binding Ep?: might this amino acid be incorporated into anantigen epitope? DVc: DENV1-4 complex; Jec: JE complex (medicallyimportant clade = JE, MVE, WN, SLE) SDM: site-directed mutagenesis

TABLE 9 Potential JEV complex- and sub-complex cross-reactive epitoperesidues from SLEV, with residues chosen for mutagenesis highlighted

*not identified as ≧ 35% SA in this particular structure/model B-f:β-factor (temperature factor) a qualitative assessment of the scale(5-60Å²). SC?: is the amino acid side chain accessible and available forantibody binding Ep?: might this amino acid be incorporated into anantigen epitope? DVc: DENV1-4 complex; Jec: JE complex (medicallyimportant clade = JE, MVE, WN, SLE) SDM: site-directed mutagenesis

TABLE 10 Inverse log₁₀ end-point titers of anti-SLEV mAbs determined bythe AG-ELISA for antigens expressed by wild-type pCB8SJ2 andcross-reactive reduced mutated constructs

Shaded block: Significantly altered endpoints relative to pCB8SJ2derived wild-type VLP antigens. Most substitutions reduced mAbreactivity, however, some mAbs reactivity increased.

TABLE 11 Inverse log₁₀ end-point titers of anti-WNV mAbs determined bythe AG-ELISA for antigens expressed by wild-type pCBWN andcross-reactive reduced mutated constructs

Shaded block: Significantly altered endpoints relative to pCB8SJ2derived wild-type VLP antigens. Most substitutions reduced mAbreactivity, however, some mAbs reactivity increased.

TABLE 12 Comparative detection of human IgM antibody by MAC-ELISA withwild type (wt-) and G106V- prototype type-specific antigens.Positive/Negative Ratios² Serum Specimen Description PRNT₉₀ ² Ref. Lab.Result VLP MAC-ELISA Infecting Virus No Class¹ SLEV WNV SLEV WNV wtG106V SLEV 1 equivocal 160 20 1.10 0.81 1.12 1.02 2 equivocal 160 201.17 1.20 1.11 1.16 3 equivocal 320 40 2.10 1.30 1.83 1.15 4 equivocal320 80 2.40 2.90 1.74 1.34 5 equivocal 320 20 8.56 8.27 3.12 1.99 6misleading 1280 160 8.27 10.8 5.40 5.09 7 misleading 1280 20 9.81 11.16.42 2.35 8 misleading 640 20 12.4 14.9 3.76 2.48 9 misleading 160 4013.0 20.3 2.02 1.47 10 misleading 1280 10 11.8 43.7 9.80 2.04 No.positive 6 6 5 1 WNV 1 positive 40 160 3.37 7.88 1.91 2.45 2 positive160 2560 1.48 5.76 3.12 4.09 3 positive 10 320 1.29 8.61 4.21 3.20 4positive 80 320 2.73 8.38 2.71 3.04 5 positive 40 2560 2.12 26.3 6.689.04 6 positive 40 1280 2.14 28.8 8.27 10.2 No. positives 1 6 4 5 ¹Serawere assigned to one of three classes; positive, equivocal, ormisleading as described in materials and methods. Assignments were basedupon previously determined P/N ratios³reported by the Diagnostics andReference Laboratory, Arbovirus Diseases Branch, Division ofVector-Borne Diseases, US Centers for Disease Control and Prevention.²PRNT₉₀, Plaque reduction neutralization test; titers represent inverse90% plaque reduction endpoints as reported by the Diagnostics andReference Laboratory, ADB, DVBID, CDC. ³Values represent ratioscalculated as described in Materials and Methods. Positive ratios ≧3.0are shown in bold ⁴Ratios reported by the Diagnostics and ReferenceLaboratory, ADB, DVBID, CDC. ⁵Ratios determined in this study comparingwild-type (wt-) WNV Ag. with prototype cross-reactivity reducedG106V-WNV Ag.

TABLE 13 Type-specific neutralizing antibody titers as determined byPRNT Plasmid DNA used for Mouse Type-specific 75% immunization¹ No. PRNTtiter² pCB8D2-2J-2-9-1 1 >128 (wt DENV-2) 2 >128 3 >128 4 64 5 >1286 >128 pCB8D2-2J-2-9-1-G106Q 1 >128 (DENV-2 + G106Q) 2 >128 3 >1284 >128 5 128 6 >128 pCBWN 1 >128 (wt WNV) 2 >128 3 >128 4 >128 5 >1286 >128 pCBWN-G106V 1 64 (WNV + G106V) 2 >128 3 16 4 >128 5 >128 6 >128¹Mice were immunized intramuscularly with 100 ug of plasmid DNA on weeks0 and 3. ²PRNT plaque reduction neutralization test, 75% neutralizationendpoint titers on mouse sera collected 6 weeks post vaccination.

1. An isolated mutant flavivirus polypeptide, comprising an amino acidsequence as shown in SEQ ID NO: 14, wherein at least one of the aminoacids at position 104, 106, 107, 126, 226, or 231 is substitutedcompared to a wild-type flavivirus polypeptide, and wherein thepolypeptide exhibits measurably reduced antibody cross-reactivity. 2.The polypeptide of claim 1, wherein the amino acid substitution isselected from the group consisting of: G₁₀₄H (SEQ ID NO: 16); G₁₀₆Q (SEQID NO: 18); L₁₀₇K (SEQ ID NO: 20); E₁₂₆A (SEQ ID NO: 22); T₂₂₆N (SEQ IDNO: 24); W₂₃₁F (SEQ ID NO: 26); W₂₃₁L (SEQ ID NO: 28); E₁₂₆A/T₂₂₆N (SEQID NO: 30); or a combination of two or more thereof.
 3. An isolatedmutant flavivirus polypeptide, comprising an amino acid sequence asshown in SEQ ID NO: 81, wherein at least one of the amino acids atposition 104, 106, or 107 is substituted compared to a wild-typeflavivirus polypeptide, and wherein the polypeptide exhibits measurablyreduced antibody cross-reactivity.
 4. The polypeptide of claim 3,wherein the amino acid substitution is selected from the groupconsisting of: G104H; G106Q (SEQ ID NO: 83); L107K; or a combination oftwo or more thereof.
 5. An isolated mutant flavivirus polypeptide,comprising an amino acid sequence as shown in SEQ ID NO: 85, wherein atleast one of the amino acids at position 104, 106, or 107 is substitutedcompared to a wild-type flavivirus polypeptide, and wherein thepolypeptide exhibits measurably reduced antibody cross-reactivity. 6.The polypeptide of claim 5, wherein the amino acid substitution isselected from the group consisting of: G104N; G106V (SEQ ID NO: 87)L107Y; or a combination of two or more thereof.
 7. An isolated nucleicacid molecule encoding a polypeptide according to claim
 1. 8. Thenucleic acid molecule of claim 7, comprising a nucleic acid sequence asshown in any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 25, 27, 29, 80,82, 84, or
 86. 9. A recombinant nucleic acid molecule, comprising aregulatory sequence operably linked to the nucleic acid molecule ofclaim
 7. 10. A cell, comprising the recombinant nucleic acid molecule ofclaim
 9. 11. The cell of claim 10, wherein the cell is a eukaryoticcell.
 12. The cell of claim 11, wherein the cell is an animal cell. 13.A virus-like particle, comprising the polypeptide of claim
 1. 14. Amethod for identifying a flavivirus cross-reactive epitope, comprising:selecting a candidate epitope using a structure-based design approach;designing a substituted epitope comprising at least one amino acidresidue substitution compared to the candidate epitope; contacting thecandidate epitope with a specific binding agent under conditions wherebya candidate epitope/specific binding agent complex can form; andcontacting the substituted epitope with the specific binding agent underthe same conditions used for candidate epitope/specific binding agentcomplex formation, wherein a candidate epitope is identified as theflavivirus cross-reactive epitope when the substituted epitope has asubstantially lower binding affinity for the specific binding agentcompared to the candidate epitope.
 15. The method of claim 14, whereinthe specific binding agent is an antibody.
 16. The method of claim 14,wherein the flavivirus cross-reactive epitope binds to a specificbinding agent that binds to at least two flaviviruses selected from thegroup consisting of dengue serotype 1 virus, dengue serotype 2 virus,dengue serotype 3 virus, dengue serotype 4 virus, yellow fever virus,Japanese encephalitis virus, St. Louis encephalitis virus, and West Nilevirus.
 17. The method of claim 14, wherein the structure-based designapproach comprises: identifying at least one conserved flavivirus aminoacid between two or more flavivirus groups or subgroups; and mapping theconserved flavivirus amino acid onto a structure of a flavivirusE-glycoprotein.
 18. The method of claim 17, wherein the flaviviruses areselected from the group consisting of dengue serotype 1 virus, dengueserotype 2 virus, dengue serotype 3 virus, dengue serotype 4 virus,yellow fever virus, Japanese encephalitis virus, St. Louis encephalitisvirus, and West Nile virus.
 19. The method of claim 17, wherein theconserved flavivirus amino acid exhibits two or more of the followingstructural characteristics: it is located in domain II of theE-glycoprotein; it is conserved across the flaviviruses; it is on theouter or lateral surface of the E-glycoprotein dimer; it has at least35% surface accessibility potential; its side chain projection isaccessible for antibody paratopes; and it has a high β-factor.
 20. Themethod of claim 14, wherein the structure-based design approachcomprises: identifying at least one conserved flavivirus amino acidbetween two or more flavivirus complexes or subcomplexes; and mappingthe conserved flavivirus amino acid onto a structure of a flavivirusE-glycoprotein.
 21. The method of claim 20, wherein the flaviviruses areselected from the group consisting of dengue serotype 1 virus, dengueserotype 2 virus, dengue serotype 3 virus, dengue serotype 4 virus,yellow fever virus, Japanese encephalitis virus, St. Louis encephalitisvirus, and West Nile virus.
 22. The method of claim 20, wherein theconserved flavivirus amino acid exhibits two or more of the followingstructural characteristics: it has at least 35% surface accessibilitypotential; it is on the outer or lateral surface of the E-glycoproteindimer; it is conserved across the flaviviruses; its side chainprojection is accessible for antibody paratopes; and it has a highβ-factor.
 23. The method of claim 14, wherein the specific binding agentis a flavivirus cross-reactive antibody.
 24. The method of claim 23,wherein the flavivirus cross-reactive antibody is selected from thegroup consisting of 4G2, 6B6C-1, 1B7-5, 10A1D-2, 4A1B-9, and 2B5B-3. 25.A composition, comprising the polypeptide of claim 1 and apharmaceutically acceptable carrier.
 26. A method of eliciting an immuneresponse against a flavivirus antigenic epitope in a subject, comprisingintroducing into the subject the composition of claim 25, therebyeliciting an immune response against a flavivirus antigenic epitope inthe subject.
 27. The method of claim 26, wherein the flavivirusantigenic epitope is selected from the group consisting of dengueserotype 1 virus, dengue serotype 2 virus, dengue serotype 3 virus,dengue serotype 4 virus, yellow fever virus, Japanese encephalitisvirus, St. Louis encephalitis virus, and West Nile virus.
 28. The methodof claim 26, wherein the subject is a mammal.
 29. A composition,comprising a nucleic acid vector, wherein the vector comprises thenucleic acid molecule of claim 7, and a pharmaceutically acceptablecarrier.
 30. A method of eliciting an immune response against aflavivirus antigenic epitope in a subject, comprising introducing intothe subject the composition of claim 29, thereby eliciting an immuneresponse against a flavivirus antigenic epitope in the subject.
 31. Themethod of claim 30, wherein the flavivirus antigenic epitope is selectedfrom the group consisting of dengue serotype 1 virus, dengue serotype 2virus, dengue serotype 3 virus, dengue serotype 4 virus, yellow fevervirus, Japanese encephalitis virus, St. Louis encephalitis virus, andWest Nile virus.
 32. The method of claim 30, wherein the subject is amammal.
 33. A method of detecting a flavivirus antibody in a sample,comprising: (a) contacting the sample with the polypeptide of claim 1under conditions whereby a polypeptide/antibody complex can form; and(b) detecting polypeptide/antibody complex formation, thereby detectinga flavivirus antibody in a sample.
 34. A method of diagnosing aflavivirus infection in a subject, comprising: contacting a sample fromthe subject with the polypeptide of claim 1 under conditions whereby anpolypeptide/antibody complex can form; and detectingpolypeptide/antibody complex formation, thereby diagnosing a flavivirusinfection in a subject.
 35. A flavivirus E-glycoprotein engineered tocomprise at least one amino acid residue substitution according to themethod of claim
 19. 36. (canceled)
 37. A flavivirus E-glycoproteinengineered to comprise at least one amino acid residue substitutionaccording to the method of claim 22.